XIII European Conference on the Spectroscopy of ... - ecsbm 2009

<strong>XIII</strong> <strong>European</strong> <strong>Conference</strong> 

on the 

Spectroscopy of Biological Molecules 

August 28 th – September 2 nd 

University of Palermo, Parco d’Orléans Campus 

Palermo, Italy 

BOOK OF ABSTRACTS 

i

ECSBM: an <strong>European</strong> Congress since 1985. 

The <strong>European</strong> <strong>Conference</strong> on the Spectroscopy of Biological Molecules (ECSBM) has been 

founded in 1985 by the researchers working in the field of optical spectroscopy applied to 

biomolecules and biosystems. The first meeting of this conference was held in Reims (France) and 

organized by the University of Reims-Champagne-Ardenne. 

Since this time, the field of activity of the conference has considerably grown up. Researchers who 

use traditional spectroscopic techniques, such as Raman scattering, IR absorption, UV/Vis 

Absorption, circular dichroism, fluorescence, magnetic resonance, X-rays and neutron scattering, as 

well as those who work on cellular and tissutal imaging attend the meetings of the conference which 

are held every two years in a different <strong>European</strong> country. Recently, particular attention has been 

devoted to the applications of biomolecular spectroscopy in the fields of biomedical imaging, anti 

cancer research, drug characterization for pharmaceutical applications, drug delivery and nanobiotechnology. 

Researchers not only from all countries of Europe attend the conference, but also 

from North Africa, Israel and Middle East countries, India, Japan and Australia, United States, 

Middle and South America. 

For its qualified attendance and for the broadness of scientific topics covered, ECSBM has become 

during the last years a leading conference located at the crossroad of three fundamental scientific 

fields: Physics, Chemistry, Biology. 

<strong>European</strong> Countries and cities where the ECSBM meetings have been held: 

1985 France (Reims) 

1987 Germany (Fribourg) 

1989 Italy (Rimini) 

1991 United Kingdom (York) 

1993 Greece (Loutraki) 

1995 France (Lille) 

1997 Spain (Madrid) 

1999 Netherlands (Enschede) 

2001 Czech Republic (Prague) 

2003 Hungary (Szeged) 

2005 Germany (Aschaffenburg) 

2007 France (Paris) 

2009 Italy (Palermo) 

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ECSBM <strong>European</strong> Committee: Scientific Council of the Congress 

Halina Abramczyk (Technical University of Lodz, Poland) abramczy@mbi-berlin.de 

Andreas Barth (Stockholm University, Sweden) barth@dbb.su.se 

Vladimir Baumruk (Charles University, Czech Republic) baumruk@karlovu.miff.cuni.cz 

Antonio Cupane (University of Palermo, Italy) cupane@fisica.unipa.it 

Manuel Dauchez (Univ.of Reims Champagne-Ardenne, France) manuel.dauchez@univ-reims.fr 

Alessandro Feis (University of Florence, Italy) feis@chim.unifi.it 

Joachim Heberle (Bielefeld University, Germany) j.heberle@fz-jjuelich.de 

Belen Hernández (BioMoCeTi, University of Paris 13, France) belen.hernandez@univ-paris13.fr 

Nerea Iza (Universidad Complutense de Madrid, Spain) nereaiza@quim.ucm.es 

Zoltan Kota (Biological Research Center, Szeged, Hungary) zkota@brc.hu 

Maria Paula Marques (University of Coimbra, Portugal) pmc@ci.uc.pt 

Dieter Naumann (Robert Koch Institute, Berlin, Germany) naumannd@rki.de 

Juan Carlos Otero (University of Malaga, Spain) jc_otero@uma.es 

Anthony Parker (Rutherford Appleton Lab. United Kingdom) a.w.parker@stfc.ac.uk 

George B. Tolstorozhev (Natl. Acad. Sci. of Belarus, Belarus) gbt@imaph.bas-net.by 

Rienk van Grondelle (Vrije Universiteit, The Netherlands) rienk@few.vu.nl 

Costantinos Varotsis (University of Crete, Greece) varotsis@edu.uoc.gr 

Local Organizing Committee 

Antonio Cupane (Chairman, DSFA Palermo, Italy) 

Grazia Cottone (DSFA Palermo, Italy) 

Daniela Giacomazza (CNR Palermo, Italy) 

Matteo Levantino (DSFA Palermo, Italy) 

Alessandro Longo (CNR Palermo, Italy) 

Giorgio Schirò (DSFA Palermo, Italy) 

Eugenio Vitrano (DSFA Palermo, Italy) 

iii

CONFERENCE PROGRAM 

August 28 th 2009 ; Friday 

15:00 – 19:00 REGISTRATION 

19:00 – 20.30 WELCOME COCKTAIL 

v

09:00 

09:30 

10:30 

11:30 

11:50 

12:15 

12:40 

13:05 

13:30 

15:00 

16:30 

OPENING CEREMONY 

August 29 th 2009 ; Saturday 

Aula Magna “Vincenzo Li Donni” 

OPENING SESSION 


Chair: Anthony W. Parker 

The tumour suppressor p53: from structure to drug discovery 

Alan FERSHT 

University of Cambridge, Cambridge, UK 

2D-IR spectroscopy of peptides and proteins 

Peter HAMM 

University of Zurich, Zurich, CH 

Coffee Break 

ADVANCED SPECTROSCOPY 1 

Complesso Polididattico Room A 

Chair: Halina Abramczyk 

Recent studies of coherent vibrational motion in 

biomolecules 

Paul M. CHAMPION 

Northeastern University, Boston, USA 

Green fluorescent protein as a matrix for the 

study of ultrafast proton transfer in proteins 

Stephen R. MEECH 

University of East Anglia, Norwich, UK 

Time-resolved diffusion and thermodynamics 

revealed spectrally hidden dynamics of 

phototropins 

Masahide TERAZIMA 

University of Kyoto, Kyoto, JP 

Ultrafast protein response in BR and 

SRII: results from isotopically labeled 

and chemically modified retinal proteins 

Rolf DILLER 

University of Kaiserslautern, 

Kaiserslautern, DE 

Lunch 

vi 

ADVANCED SPECTROSCOPY 2 

Complesso Polididattico Room B 

Chair: Andreas Barth 

Laser amplifier for spectroscopy of protonated 

peptides 

Oleg V. BOYARKIN 

École Polytechnique Fédérale , Lausanne, CH 

Spectroscopic and modeling studies of 

biomolecular adsorption on solid surfaces 

Dennis K. HORE 

University of Victoria, Victoria, CA 

Structural analysis of macromolecular 

complexes by isotope-edited FTIR spectroscopy 

Suren A. TATULIAN 

University of Central Florida, Orlando, USA 

Exciton dynamics and energy disorder in 

photosynthetic light-harvesting complexes 

Silvia VOLKER 

University of Leiden, Leiden, NL 

Poster Session A: Magnetic Resonance; Multidimensional Spectroscopy; Neutron 

Scattering; Optical Spectroscopy; Synchrotron and XFEL Spectroscopy 

Coffee Break

16:50 

17:50 

18:15 

18:40 

19:05 

PROTEIN DYNAMICS 1 


Chair: Lorenzo Cordone 

Protein dynamics and functionality: the 

role of hydration shell waters 

Joel FRIEDMAN 

AECOM, New York, USA 

Fluorescence and Infrared Cross- 

Correlation spectroscopy: a new tool in 

analysing protein conformational 

coupling 

Karim FAHMY 

Forschungszentrum, Dresden, DE 

Shape of the carbon monoxide infrared 

absorption band of carboxyheme proteins as a 

probe of the protein anharmonicity 

Solomon S. STAVROV 

University of Tel-Aviv, Tel-Aviv, IL 

Low-frequency dynamics of Bacteriorhodopsin 

studied by terahertz time-domain spectroscopy; 

relation with its function 

Keisuke TOMINAGA 

University of Kobe, Kobe, JP 

Low temperature FTIR spectroscopy reveals new 

insights on the pH-dependent proton pathway of 

Proteorhodopsin 

Gabriela SCHÄFER 

Goethe-University, Frankfurt, DE 

vii 

MAGNETIC RESONANCE 


Chair: Alessandro Feis 

EPR studies of membrane protein 

structure and folding 

Gunnar JESCHKE 

ETH, Zurich, CH 

Time resolved EPR investigation on oxygen and 

temperature effects on a synthetic eumelanin 

Fosca CONTI 

University of Padova, Padova, IT 

Oligonucleotide-based chemical nucleases: 

opportunities and challenges in RNA targeting 

Steven M. MILES 

University of Manchester, Manchester, UK 

Molecular stress in biological membranes 

measured by solid-state NMR spectroscopy 

Horia I. PETRACHE 

Purdue University, Indianapolis, USA 

Self-assembled ion channels probed by nitroxide 

spin labels and PELDOR spectroscopy 

Jan RAAP 

University of Leiden, Leiden, NL

09:00 

10:00 

10:25 

10:50 

11:10 

12:10 

12:35 

13:00 

13:30 

15:00 

16:30 

August 30 th 2009 ; Sunday 

FTIR, X-RAY AND NEUTRON SCATTERING STUDIES ON PROTEINS AND PEPTIDES 


Chairs: Antonio Cupane – Rienk van Grondelle 

Protein structural dynamics visualized by time-resolved X-ray crystallography and 

liquidography 

Hyotcherl IHEE 

KAIST, Daejeon, RoK 

Tracking structural changes in solution with 100 ps time-resolved X-ray scattering 

Marco CAMMARATA 

ESRF, Grenoble, FR 

Proteins in action monitored by time-resolved FTIR spectroscopy 

Klaus GERWERT 

Ruhr University, Bochum, DE 


Neutron scattering studies of low-temperature and high-temperature dynamic 

crossovers in lysozyme hydration water and their effect on the conformational 

dynamics of the protein 

Sow-Hsin CHEN 

MIT, Cambridge, USA 

Sponsored by “Spectroscopy – Biomedical Applications” - IOS Press 

Dynamic crossovers and quantum effects in protein hydration water 

Fabio BRUNI 

University of Rome 3, Rome, IT 

Folding dynamics of peptides studied by temperature-jump infrared-spectroscopy 

Karin HAUSER 

University of Frankfurt , Frankfurt, DE 

Photoemission and the shape of free amino acids 

Vitaliy FEYER 

Synchrotron Trieste, Basovizza, IT 

Lunch 

Poster Session A: Magnetic Resonance; Multidimensional Spectroscopy; Neutron 

Scattering; Optical Spectroscopy; Synchrotron and XFEL Spectroscopy 


viii

16:50 

17:50 

18:15 

18:40 

19:05 

PROTEIN DYNAMICS 2 


Chair: Balazs Szalontai 

Hydrogen bonding properties of 

amorphous water-saccharide matrixes 

and bioprotection 

Lorenzo CORDONE 

University of Palermo, Palermo, IT 

Modulation of the conformation of cytochrome c 

oxidase from Paracoccus denitrificans by activesite 

mutations 

Denis L. ROUSSEAU 

AECOM, New York, USA 

A new method to monitor ligand binding to 

proteins, amide I band simulations and the 

infrared spectrum of acetyl phosphate 

Andreas BARTH 

University of Stockholm, Stockholm, SE 

The regulation of the formation of 

cytoskeletal protein complexes by actinbinding 

proteins 

Miklos NYITRAI 

University of Pécs, Pécs, HU 

Study of dynamic properties of reconstituted 

myelin sheath 

Francesca NATALI 

CNR/ILL, Grenoble, FR 

ix 

OPTICAL SPECTROSCOPY 1 


Chair: Oleksandr Slobodyanyuk 

dSTORM: super-resolution imaging with 

small organic fluorophores 

Markus SAUER 

University of Bielefeld, Bielefeld, DE 

Disulfide chromophore and its optical activity 

Vladimir BAUMRUK 

Charles University, Prague, CZ 

Ultrafast processes in photosynthesis 

Rienk van GRONDELLE 

Vrije Universiteit, Amsterdam, NL 

Steady state and time-resolved Fluorescence 

measurements of the Green Fluorescent 

Variants T203V and T203V/S205V 

Dan HUPPERT 

University of Tel-Aviv, Tel-Aviv, IL 

The influence of activator on conformational 

changes in human platelet Integrin αIIbβ3 

Tia E. KEYES 

Dublin City University, Dublin, IE

09:00 

10:00 

10:30 

11:00 

11:20 

11:45 

12:10 

12:35 

13:00 

13:25 

15:00 

16:30 

August 31 st 2009 ; Monday 

PROTEIN FOLDING AND AGGREGATION 


Chairs: Pier Luigi San Biagio – Zoltan Kota 

Polyglutamine and neurodegeneration: a 

structural approach 

Laura MASINO 

NIMR, London, UK 

Unfolded and (self)-aggregated states of alanine 

based peptides 

Reinhard SCHWEITZER-STENNER 

Drexel University, Philadelphia, USA 

A novel approach to multiscale measurements in 

biological materials: silk as a model system 

Cedric DICKO 

University of Oxford, Oxford, UK 


UV-Vis and FT-IR spectra of ultraviolet 

irradiated collagen in the presence of antioxidant 

ascorbic acid 

Ketevan JARIASHVILI 

Tbilisi State University, Tbilisi, GE 

Equilibrium spectroscopic studies of aromaticaromatic 

interaction and pH effect on trpzip βhairpin 

stability 

Timothy A. KEIDERLING 

University of Illinois at Chicago, Chicago, USA 

Denaturation of proteins with beta-barrel 

topology induced by guanidine hydrochloride 

Olesya STEPANENKO 

RAS, Saint-Petersburg, RU 

Determination of conformational changes during 

aggregation of the amyloid-beta peptide by 

attenuated total reflection-Fourier transform 

infrared spectroscopy 

Rabia SARROUKH 

Université Libre de Bruxelles, Bruxelles, BE 

A 2DCOS study of the effect of radiation on 

TGase activity 

José Luis ARRONDO 

Universidad del País Vasco, Bilbao, ES 

Lunch 

x 

BIOMEDICAL APPLICATIONS 1 


Chairs: Maria P. Sevilla – Roger Bisby 

Will biophysical studies of neuroglobin 

provide clues for novel therapies of 

neuronal death and degeneration? 

Beatrice VALLONE 

University “La Sapienza”, Rome, IT 

Biomedical application of Mössbauer 

spectroscopy with high velocity resolution: 

revealing of small variations 

Michael I. OSHTRAKH 

Ural State Technical Univ., Ekaterinburg, RU 

Raman markers of cancer. Ultrafast dynamics of 

carotenoids and lipids 

Halina ABRAMCZYK 

Technical University of Lodz, Lodz, PL 

Far-IR synchrotron and low-wavenumber 

Raman studies of proteins and protein/water 

interactions. From model system to animal and 

human skin 

Ole F. NIELSEN 

University of Copenhagen, Copenhagen, DK 

Histopathological characterization of skin 

cancers using infrared micro-imaging 

Elodie LY 

Univ. Reims-Champagne Ardenne, Reims, FR 

Investigation of the Raman spectra of hemozoin 

Torsten FROSCH 

University of Jena, Jena, DE 

Automated atmospheric pressure ionization 

mass spectrometry imaging platform 

Vladimir HAVLICECK 

ASCR, Prague, CZ 

Solubilization and determination of 

Ketoconazole in micellar solution of 

polyethylene glycol 400 

Neeti NEMA 

Hari Singh Gour University, IN 

Poster Session B : Biomedical Applications; Dielectric Spectroscopy; DNA & RNA; 

Nanotechnology … ; Protein Dynamics; Protein Folding and Aggregation; Single Molecule 

and Single Cell Spectroscopy

17:00 Guided tours of: 

Free afternoon 

- Palermo Botanical Garden, Via Lincoln 2; http://ortobotanico.palermo.it/ 

- Palazzo Steri with Inquisition Jails, Piazza Marina 61 (max. 100 persons) 

20:30 Social Dinner: Palazzo Butera, Via Butera, 8 

xi

09:00 

10:00 

10:25 

10:50 

September 1 st 2009 ; Tuesday 

OPTICAL SPECTROSCOPY 2 


Chairs: V. K. Rastogi – Belen Hernandez - Illera 

Heme pocket structural properties of 

bacterial truncated hemoglobins as 

revealed by resonance Raman 

spectroscopy 

Giulietta SMULEVICH 

University of Florence, Florence, IT 

Primary events in the photo-dissociation of oxyhemoglobin 

Atsushi YABUSHITA 

National Chiao-Tung Univ., Hsichu, Taiwan 

UV Resonance Raman spectroscopic study of 

tyrosine in the TTR(105-115) peptide 

Sophia C. HAYES 

University of Cyprus, Nicosia, CY 

Rapid-scan/step-scan FTIR difference 

spectroscopy applied to photosynthetic 

reactions: new insights by multiple experiment 

design and tailored multivariate analysis 

techniques 

Alberto MEZZETTI 

University of Science and Technology, Lille, FR 

11:15 Coffee Break 

11:30 

11:50 

12:10 

12:30 

12:50 

Does the disulfide bridge have an effect 

on the conformational properties of the 

peptide hormone somatostatin-14? 

Mahmoud GHOMI 

University of Paris 13, Bobigny, FR 

Model based pre-processing in biospectroscopy 

Nils K. AFSETH 

Nofima-Mat, Ås, NO 

Stemness of stem cells as determined by 

confocal Raman microspectroscopy 

Vishnu V. PULLY 

University of Twente, Enschede, NL 

Tautomerism in 5-Bromouracil: relationships 

with other 5-haloderivatives and effect of the 

microhydration 

Vinod K. RASTOGI 

CCS University, Meerut, IN 

Activity of upper electron-excited states in 

coelenterate bioluminescence 

Nadezda BELOGUROVA 

RAS - Siberian Branch, Krasnoyarsk, RU 

xii 

NANOTECHNOLOGY 

SINGLE MOLECULE SPECTROSCOPY 


Chairs: Jose V. Garcia Ramos – Nerea Iza 

Atomic force spectroscopic investigation 

of biorecognition events 

Salvatore CANNISTRARO 

University of Tuscia, Viterbo, IT 

Observing proteins as single molecules 

encapsulated in surface- tethered polymeric 

nanocontainers 

Joerg FITTER 

Forschungszentrum, Jülich, DE 

Three dimensional collagen gels as a cell culture 

model for the study of living cells by Raman 

spectroscopy 

Franck BONNIER 

Dublin Institute of Technology, Dublin, IE 

Gold nanoparticles for protein detection assays 

Giuseppe CHIRICO 

University of Milano “Bicocca”, Milano, IT 

Data processing in FTIR imaging of cells and 

tissues: towards protein secondary structure 

imaging 

Erik GOORMAGHTIGH 

Université Libre de Bruxelles, Bruxelles, BE 

Nanoparticle-based SERS for biodiagnostic 

sensing 

Janina KNEIPP 

Humboldt University, Berlin, DE 

Tip-enhanced and surface-enhanced Raman 

spectroscopy of biological molecules on 

structured metallic surfaces 

Laura E. HENNEMANN 

University of Tübingen, Tübingen, DE 

SERS microscopy: improved nanoparticle 

probes and their application in tissue diagnostics 

Max SCHÜTZ 

University of Osnabrück , Osnabrück, DE 

Surface enhanced Raman scattering for tissue 

diagnostic 

Simona CÎNTĂ PÎNZARU 

Babes-Bolyai University, Cluj-Napoca, RO

13:10 

13:30 

15:00 

FTIR microspectroscopy (MSP) for detection 

and identification of fungal phytopathogenes 

Ahmad SALMAN 

SCE, Beer-Sheva, IL 

Lunch 

xiii 

A new approach for online-monitoring of drugs 

in complex matrices with Surface Enhanced 

Raman Spectroscopy 

Anne MAERZ 

Friedrich Schiller University, Jena, DE 

Poster Session B : Biomedical Applications; Dielectric Spectroscopy; DNA & RNA; 

Nanotechnology … ; Protein Dynamics; Protein Folding and Aggregation; Single Molecule 

and Single Cell Spectroscopy 


16:50 

17:15 

17:40 

18:05 

18:30 

BIOMEDICAL APPLICATIONS 2 


Chair: Vladimir Baumruk 

Towards the instantaneous quantitative 

fluoroimaging drugs determination in body 

fluids with no added reagents 

Abraham H. PAROLA 

Ben Gurion University, Beer-Sheva, IL 

Near-Infrared fluorophores for biomolecule 

characterization and imaging applications 

Gabor PATONAY 

Georgia State University, Atlanta, USA 

Examination of IgG diffusion in controlled pore 

glass protein affinity media with Confocal 

Raman Spectroscopy 

Nanying BIAN 

Millipore Corp., Bedford, USA 

Study of melanoma cell lines invasiveness using 

Raman microspectroscopy 

Olivier PIOT 

Univ. of Reims-Champagne Ardenne, Reims, FR 

Synchrotron based FTIR Microscopy of single 

stained lung cells: applications in cancer 

diagnosis 

Josep SULE’-SUSO 

University of Keele, UK 

DNA&RNA / DIELECTRIC 

SPECTROSCOPY 


Chair: Shaul Mordechai 

Excitation of cytosine-rich nucleic acids as seen 

through time-resolved infrared spectroscopy 

Anthony W. PARKER 

Rutherford Appleton Laboratory, Chilton, UK 

Emission lifetime study of fluorescence probes 

based on G-quadruplex oligonucleotides endlabeled 

with pyrene moieties 

Bernard JUSKOWIAK 

Mickiewicz University, Poznan, PL 

DNA photodamage: study of thyminephotodimerisation 

in a locked thymine 

dinucleotide 

Wolfgang J. SCHREIER 

LMU, München, DE 

C`-terminal domain of nonhistone protein 

HMGB1 as a modulator of HMGB1–DNA 

structural interactions 

Elena CHIKHIRZHINA 

RAS, Saint-Petersburg, RU 

Microwave hydration measurements of the 

unfolding in aqueous systems of proteins and 

synthetic proteinlike polymers 

Mikhail M. VOROB’EV 

RAS, Moscow, RU

09:00 

10:00 

10:25 

10:50 

September 2 nd 2009 ; Wednesday 

THEORETICAL/COMPUTATIONAL METHODS 


Chair: Mario Pio Marzocchi 

Theoretical search for molecular mechanisms of photostability of building blocks 

of life 

Andrzej SOBOLEWSKI 

Polish Academy of Sciences, Warsaw, PL 

Computing vibrational spectra of biomolecules by mixed Quantum Mechanics / Molecular 

Mechanics 

Leonardo GUIDONI 

University of L’Aquila, Monteluco di Roio, IT 

A statistical model for protein translocation across nanopores 

Fabio CECCONI 

CNR – INFM, Rome, IT 

Respiratory proteins: breathing motions revealed by Molecular Dynamics simulations 

Arturo ROBERTAZZI 

University of Cagliari, Cagliari, IT 


11:30 

Closing Lecture 

Water in narrow pores 

Christoph DELLAGO 

University of Vienna, Vienna, Austria 

12:30 STUDENT PRIZES AWARDING AND CLOSING CEREMONY 

xiv

Palermo, August 28 – September 2, 2009 13 th ECSBM −−−− Book of Abstracts 

INVITED LECTURES 

1


2


The Tumour Suppressor p53: from structure to drug discovery 

ALAN FERSHT 

Cambridge University and MRC Centre for Protein Engineering 

Hills Road, Cambridge, CB2 0QH, UK 

p53 is directly inactivated by mutation in some 50% of human cancers. Some 30-40% of the 

mutations simply lower the stability of the core domain so it melts close to or below body 

temperature. We have shown in principle that it is possible to reactivate p53 by small molecules 

that bind to and stabilise it. To understand further the structure of the protein and hence the 

rational design of drugs, we are solving its structure at high resolution. We are faced with twin 

problems: the tetrameric protein consists of 1572 residues, some of which are in well-structured 

domains but others are intrinsically disordered or natively unfolded; and the important core 

domain is intrinsically unstable and not well suited to systematic study. We have solved the 

structure of the core domain in solution by state-of-the-art NMR methods and found structural 

reasons for its instability. We have engineered a more stable variant, which is biologically active 

and have solved the crystal structures of oncogenic mutants in this framework. We solved the 

quaternary structure of the full-length tetrameric complex by combining high-resolution structural 

information on the folded individual domains with NMR, small angle x-ray scattering and electron 

microscopy, which should be a paradigm for solving other complex proteins that are involved in 

the cell cycle. We are refining these structures to high resolution, and studying their complexes 

with partner proteins using a further mix of structural methods. The mutation Y220C occurs in 

about 70,000 to 80,000 new cases of cancer per annum. The mutant is highly destabilized and 

denatures rapidly at body temperature. Our structural studies revealed that the mutation forms a 

surface cavity that appears a prime target for small molecules to bind to and stabilise the protein. 

In silico design identified a series of molecules that might bind in the cavity. We screened those 

and the second and third generation derivatives and found small compounds of drug-like 

properties that raise the melting temperature of the mutants and restore its activity. 

3


2D-IR spectroscopy of peptides and proteins 

PETER HAMM 

University of Zürich, Institute of Physical Chemistry 

Winterthurerstr. 190, 8057 Zürich, Switzerland 

We have recently developed the method of transient 2D-IR spectroscopy, i.e. the 2D-IR 

spectroscopy of a transient species during a photochemical reaction. 2D-IR spectroscopy carries 

significant structural information through the existence of crosspeaks which report on local 

contacts in a molecule. At the same time, it takes one picosecond to measure a 2D-IR spectrum, 

which opens the possibility to measure snapshot structures during the course of a photochemical 

reaction. Transient 2D-IR spectroscopy combines ultrafast time resolution with appreciable 

structure resolution. I will report on various examples of photo-switchable peptides whose folding 

transition is studied by this new technique. 

4


Protein dynamics and functionality: the role of 

hydration shell waters 

JOEL M. FRIEDMAN 

Dept of Physiology and Biophysics, Albert Einstein College of Medicine, 

Bronx, New York USA 10461 

It has become fully apparent that protein functionality requires that the protein be dynamically 

active and that solvent motions are needed for activation of functionally important protein 

dynamics. In attempting to study the interplay between dynamics and functionality one is 

confronted with the complication that there is myriad of different types of dynamics displayed by 

proteins. How to organize these dynamics into a meaningful and workable scheme is a challenge. 

One promising approach is based on Frauenfelder and coworkers solvent slaving concept which 

defines a hierarchy of protein dynamics based on which category of solvent motion controls the 

enthalpic barrier associated with specific conformational fluctuations and relaxations. This concept 

dictates that an overall approach to the study of the dynamical basis for protein functionality 

include four elements: function, protein dynamics, solvent motions and their interdependence. The 

presentation will describe how we have used sol-gel and glass matrices in combination with an 

extension of the solvent slaving concept in the form our proposed Protein Dynamic State Model to 

link functionality, conformational dynamics and solvent properties. Additionally we will present 

advances in the use of several spectroscopic tools including vibronic side band spectroscopy to 

probe the response of hydration shell waters to added osmolytes and the correlated impact on 

protein properties. A general picture based on these studies will be presented that links hydration 

shell water properties, dynamics and functionality. 

5


EPR studies of membrane protein structure and folding 

G. JESCHKE 1 , D. HILGER 2 , H. JUNG 2 , E. PADAN 3 , Y. POLYHACH, 1 A. VOLKOV 4 , C. DOCKTER 5 AND 

H. PAULSEN 5 

1. Lab. Phys. Chem., ETH Zürich, Wolfgang-Pauli-Str. 10, Zürich, CH- 

8093, Switzerland 

2. Department Biologie I, Microbiology, LMU München, Grosshaderner 

Strasse 2-4, D-82152 Planegg-Martinsried, Germany 

3. Hebrew University of Jerusalem, Alexander Silberman Institute of Life 

Sciences, IL-91904 Jerusalem, Israel 

4. MPI for Polymer Research, Ackermannweg 10, D-55021 Mainz, 

Germany 

5. Institute for General Botany, Johannes-Gutenberg University Mainz, 

Müllerweg 6, D-55099 Mainz, Germany 

The function of many membrane proteins is related to structural transitions induced by subtle 

changes in the environment. In this situation studies of crystalline samples, which are in itself hard 

to obtain, cannot provide the full information on the structure-dynamics-function relationship. At 

the same time there is a lack of techniques that can provide high-resolution structural information 

in non-crystalline environments, in particular for medium-sized and large α-helical membrane 

proteins, where NMR techniques run into problems. By combining site-directed spin labeling [1] 

with pulsed EPR techniques [2] it is possible to obtain information even on disordered states 

encountered before or during folding or disordered domains that are missing in crystal structures. 

This is demonstrated for major plant light harvesting complex IIb [3,4]. In particular, structurally 

resolved information on folding kinetics can be obtained by following changes in water 

accessibility of a selected residue with electron spin echo envelope modulation (ESEEM) 

spectroscopy and changes in distances between two selected sites by double electron electron 

resonance (DEER) experiments. In most cases, resolution will be moderate due to the size and 

conformational distribution of the spin label. However, high-resolution structures of protein 

complexes (or oligomers) can be obtained from DEER data if structures with atomic resolution are 

known for the constituents (or monomer) [5]. On the 54.3 kDa Na +/proline symporter in liposomes 

it is demonstrated that the shape of a transmembrane domain at moderate resolution can be 

obtained “from scratch”, i.e. without regress to any previous structural information at similar or 

higher resolution [6]. 

References 

[1] W. L. Hubbell, D.S. Cafiso, C. Altenbach, Nat. Struct. Biol. 7, 735-739 (2000). 

[2] A. Schweiger, G. Jeschke, Principles of pulse electron paramagnetic resonance, Oxford: Oxford University Press, 

(2001). 

[3] G. Jeschke, A. Bender, T. Schweikardt, G. Panek, H. Decker, H. Paulsen, J. Biol. Chem. 280, 18623-18630 

(2005). 

[4] A. Volkov, C. Dockter, T. Bund, H. Paulsen, G. Jeschke, Biophys. J. 96, 1124-1141 (2009). 

[5] D. Hilger, Ye. Polyhach, E. Padan, H. Jung, G. Jeschke, Biophys. J. 93, 3675-3683 (2007). 

[6] D. Hilger, Y. Polyhach, H. Jung, G. Jeschke, Biophys. J. 96, 217-225 (2009). 

6


Protein structural dynamics visualized by time-resolved 

X-ray crystallography and liquidography 

H. IHEE 

Center for Time-Resolved Diffraction, Department of Chemistry, KAIST, 

305-701, KOREA hyotcherl.ihee@kaist.ac.kr 

The principle, experimental technique, data analysis, and applications of time-resolved X-ray 

diffraction and scattering to study spatiotemporal reaction dynamics of proteins in single crystals 

and solutions will be presented. X-ray crystallography, the major structural tool to determine 3D 

structures of proteins, can be extended to time-resolved X-ray crystallography with a laserexcitation 

and X-ray-probe scheme, and all the atomic positions in a protein can be tracked during 

their biological function. A study using time-resolved X-ray crystallography will be presented for 

the photocycle of the photoactive yellow protein (PYP). Although time-resolved X-ray 

crystallography has realized the 3D visualization of protein structural dynamics, its applicability 

has been limited to a few model systems with reversible photocycles due to the stringent 

prerequisites such as highly-ordered and radiation-resistant single crystals. More importantly, 

crystal packing constraints might hinder biologically relevant motions, and it simply cannot be 

used to study irreversible reactions such as protein folding. These problems can be overcome by 

applying time-resolved X-ray diffraction directly to protein solutions rather than protein single 

crystals and then capturing transient molecular structures in one-dimension. To emphasize that 

structural information can be obtained from the liquid phase, this time-resolved X-ray solution 

scattering technique is named time-resolved X-ray liquidography in analogy to time-resolved Xray 

crystallography where the structural information of reaction intermediates is obtained from 

the crystalline phase. Time-resolved X-ray liquidography permits us to investigate the 

tertiary/quaternary conformational changes of human hemoglobin triggered by laser induced 

ligand photolysis in nearly physiological conditions. Data on optically induced tertiary relaxations 

of myoglobin and refolding of cytochrome c are also reported to illustrate the wide applicability of 

this technique. In addition the photocycle of PYP which has been extensively investigated with 

time-resolved X-ray crystallography has been also studied with time-resolved X-ray liquidography 

so that a direct comparison between liquidography and crystallography is possible. By providing 

insights into the structural dynamics of proteins functioning in their natural environment, timeresolved 

X-ray liquidography complements and extends results obtained with time-resolved 

spectroscopy and X-ray crystallography. 

References 

[1] Ihee, H., Acc. Chem. Res., 42, 356-366 (2009). 

[2] Cammarata, M. et al., Nature Methods, 5, 881 (2008) 

[3] Lee, J. H. et al., J. Am. Chem. Soc., 130, 5834 (2008) 

[4] Kong, Q. et al., Angew. Chem. Int. Ed., 47, 5550 (2008) 

[5] Lee, J. H. et al., Angew. Chem. Int. Ed., 47, 1047 (2008) 

[6] Kong, Q. et al., J. Am. Chem. Soc., 129, 13584 (2007) 

[7] Kim, T. K. et al., Proc. Natl. Acad. Sci., 103, 9410 (2006) 

[8] Lee, J. H. et al., J. Chem. Phys., 125, 174504 (2006) 

[9] Ihee, H. et al., Science, 309, 1223 (2005) 

7


Neutron scattering studies of low-temperature and 

high-temperature dynamic crossover in lysozyme 

hydration water and their effect on the conformational 

dynamics of the protein 

SOW HSIN CHEN 

Department of Nuclear Science and Engineering, Massachusetts Institute 

of Technology, Cambridge, MA, USA (sowhsin@mit.edu) 

We study the dynamics of hydration water on lysozyme powder using Elastic, Quasi-Elastic and 

Inelastic Neutron Scattering in the temperature range from 180 K to 380 K, and in the pressure 

range from 1 bar to 1600 bars. We found well-defined dynamic crossover phenomena in the 

hydrogen atom mean square displacement and the translational relaxation time of water 

molecules. The high temperature crossover occurs at TD = 345 ± 5 K, which coincides with the 

reversible denaturation temperature of lysozyme [1]. The low temperature crossover occurs at TL = 

225 ± 5 K, which coincides with the protein dynamic transition (or so-called glass transition) 

temperature [2]. Upon applying pressure, the low-T crossover TL(P) tracks the Widom line in the 

(T,P) plane, emanating from the hypothetical existence of the liquid-liquid critical point at PC = 

1550 ± 50 bars and TC = 200 ± 5 K [3]. This suggests that the dynamics and thermodynamics of 

protein hydration water are strongly coupled to the dynamics of the protein, in such a way that the 

two crossover temperatures TL and TD define the temperature interval of the significant biological 

activity of the protein. We also performed a high-resolution inelastic x-ray scattering study of 

intra-protein phonons in hydrated lysozyme and BSA powders, above and below the low-T 

crossover temperature. We identify a significant softening and an increase in population of 

phononlike collective motions in an intermediate q-range above the dynamic transition 

temperature of the proteins [4]. 

References 

[1] Y. Zhang, M. Lagi, D. Liu, F. Mallamace, E. Fratini, P. Baglioni, E. Mamontov, M. Hagen, and S.-H. Chen, 

“Observation of high-temperature dynamic crossover in protein hydration water and its relation to reversible 

denaturation of lysozyme”, J. Chem. Phys. 130, 135101 (2009) 

[2] S.-H. Chen, L. Liu, E. Fratini, P. Baglioni, A. Faraone, and E. Mamontov, “Observation of fragile-to-strong dynamic 

crossover in protein hydration water,” Proc. Nat. Acad. Sci. USA 103, 9012-9016 (2006). 

[3] X.-Q. Chu, A. Faraone, C. Kim, E. Fratini, P. Baglioni, J. B. Leao and S.-H. Chen “Proteins remain soft at lower 

temperatures under pressure” J. Phys. Chem B (Letter) 113, 5001 (2009). 

[4] D. Liu, X.-Q. Chu, M. Lagi, Y. Zhang, E. Fratini, P. Baglioni, A. Alatas, A. Said, E. Alp and S.-H. Chen “Studies of 

Phonon-like Low-Energy Excitations of Protein Molecules by Inelastic Xray Scattering”, Phys. Rev. Lett. 101, 

135501 (2008) 

8


Hydrogen Bonding Properties of Amorphous low-Water 

protein-Saccharide Matrixes and Bioprotection 

LORENZO CORDONE 

Dipartimento di Scienze Fisiche ed Astronomiche, Università di 

Palermo, Via Archirafi 36 I-90123 Palermo, Italy 

cordone@fisica.unipa.it 

Glassy matrices of saccharides exhibit an outstanding ability to protect biological structures 

against adverse environmental conditions, such as freezing, heating, and dehydration [1,2]. 

Saccharides are employed by several organisms that can long survive under extreme drought and 

high temperature, entering a state of suspended metabolism (anhydrobiosis), which is preceded by 

a massive synthesis of specific carbohydrates [1,2]. Among sugars, the disaccharide trehalose 

appears to be the most effective protectant [1,3,4]. In this communication will be presented a set of 

measurements, performed with different experimental techniques, which concur in suggesting 

how a relevant role, in determining the difference in bioprotection among different saccharides, is 

played by the hydrogen bonding properties of low water amorphous saccharide matrixes. 

References 

[1] J. H. Crowe, J. F Carpenter, and L. M. Crowe, Annu. ReV. Physiol., 60, 73, 1998 

[2] L. M. Crowe, Comp. Biochem. Physiol. A., 132, 505, 2002 

[3] J. H.; Crowe, L. M. Crowe, S. A., Jackson, Arch. Biochem. Biophys., 220, 477, 1983 

[4] M. Uritani, M. Takai, K. Yoshinaga, J. Biochem., 117, 774, 1995, 

9


dSTORM: Super-resolution imaging 

with small organic fluorophores 

MARKUS SAUER 

Applied Laser Physics and Laser Spectroscopy and Bielefeld Institute for 

Biophysics and Nanoscience, Bielefeld University, Universitätsstrasse 25, 

33615 Bielefeld, Germany 

We introduce a general approach for multicolor super-resolution fluorescence imaging based on 

photoswitching of standard small organic fluorophores. Photoswitching of organic rhodamine and 

oxazine fluorophores, i.e. the reversible transition from a fluorescent to a non-fluorescent state in 

aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with 

thiol compounds and repopulation of the singlet ground state by reaction with molecular oxygen. 

We unravel the underlying switching mechanism and demonstrate super-resolution imaging with 

different commercially available organic fluorophores. Furthermore, we provide evidence that the 

method can be advantageously used for live cell imaging with ~ 20 nm optical resolution. 

10


Polyglutamine and neurodegeneration: 

a structural approach 

L. MASINO 

Division of Molecular Structure - MRC National Institute for Medical Research 

London - UK 

Expansion of unstable CAG trinucleotide repeats coding for polyglutamine is the cause of at least 

nine inherited neurodegenerative disorders, including Huntington's disease and several 

spinocerebellar ataxias. Protein misfolding and aggregation seem to be critical steps in the 

pathogenesis of these disorders. The structural characterisation of the proteins carrying 

polyglutamine stretches is therefore an essential prerequisite for understanding disease 

mechanisms and designing effective therapeutic strategies. Using a wide range of biophysical 

methods, including optical and NMR spectroscopy, EM, and AFM, we have investigated the 

structural properties of a polyglutamine model system and of the protein ataxin-3, elucidating 

their domain architecture, aggregation mechanisms, target interactions and function. By showing 

that ataxin-3 is a cysteine protease that cleaves poly-ubiquitin chains and is involved in the 

ubiquitin-proteasome pathway, we have shed light on the role of ubiquitin binding on ataxin-3's 

aggregation pathways. 

11


Will biophysical studies of neuroglobin provide clues 

for novel therapies of neuronal death and degeneration? 

B. VALLONE 1 , C. ARDICCIONI 1 , T. MOSCHETTI 1 , C. RONDINELLI 1 , A. GIUFFRÈ 3 , A. ARCOVITO 2 

AND M. BRUNORI 1 

1. Dept. of Biochemical Sciences, University of Rome "La Sapienza", P.le 

A. Moro, 5, 00185, Rome, Italy. 

2. Institue of Biochemistry and Clinical Biochemistry, Catholic 

University of Rome, L.go F.Vito, 00168, Rome Italy 

3. IBPM of CNR c/o University of Rome "La Sapienza, P.le A. Moro, 5, 

00185, Rome, Italy. 

Neuroglobin (Ngb), a heme protein expressed in the Central Nervous System of vertebrates, was 

shown in vivo and in vitro to counteract the effects of ischemia and the onset of Alzheimer disease 

[1]. Moreover association was shown between Ngb and the cellular prion protein (prPc) and 

between one of the subunits (G�i) of heterorimeric G-proteins. The overexpression of neuroglobin 

has beneficial effects also upon hypoxia of cardiac tissue. We have determined the threedimensional 

structure of liganded and unliganded Ngb [2,3] and characterized its reaction with 

radicals such as NO [4]. The structural dynamics of Ngb was analysed by molecular dynamics and, 

experimentally, by microspectrophotometry in crystals, XANES [5], and stopped-flow kinetics. We 

have confirmed the specific interaction of Ngb with PrPc. We are currently analyzing this complex 

by using specifically designed bio-active peptides from PrPc. The main goal is the determination of 

the molecular mechanism of neuroprotection exerted by neuroglobin, by rigorous structural and 

biophysical characterization of the system and implementation of this information in cell biology 

experiments. We are exspressing proteins that could take part in a signalling complex and 

engineering mutants of neuroglobin with altered properties (ligand affinity, reactivity towards 

radicals, complex formation) and expressing them in neuronal cells in culture to evaluate their 

effect on functions. We will determine the three-dimensional structure of complexes of Ngb and 

another partner in signalling networks (PrPc and G-alpha-i) by crystallography and X-ray 

scattering. The mechanism of neuroprotection by neuroglobin is still unclear, and its 

characterization may pave the way to novel strategies for the treatment and prevention of 

neuronal death due to ischemia and neurodenerative diseases. Our approach will yield structural 

information at the detail necessary for the rational design of new drug leads. 

Fig. 1. The structure of neuroglobin with xenon atoms docked into 

cavities provides a putative ligand migration pathway. 

12


Heme pocket structural properties of bacterial truncated 

hemoglobins as revealed by resonance Raman spectroscopy 

A. FEIS 1, E. DROGHETTI 1, B.D. HOWES 1, F.P. NICOLETTI 1, A. BOFFI 2, C. VERDE 3 AND G. SMULEVICH 1 

1. Dept. of Chemistry, Università di Firenze, Via della Lastruccia 3, Firenze, I-50019, Italy 

2. Dept. of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, University of Rome 

“La Sapienza”, Piazzale Aldo Moro 5, I-00185 Rome, Italy 

3. Institute of Protein Biochemistry, CNR, Via Pietro Castellino 111, I-80131 Naples, Italy 

Truncated hemoglobins (trHbs) are a family of small oxygen binding proteins, widely distributed 

among bacteria, protozoa and plants [1, 2], characterized by i) amino acid sequence which is 20–40 

residues shorter than animal Hbs; ii) unusual structural fold, and iii) a remarkable variability in 

the nature of the heme pocket residues in the distal side. They are divided into three groups that 

share less than 30% sequence similarity with each other [1]. Group II is the most populated of the 

three, and is characterized by the presence of a Trp residue (G8 position) in the heme distal pocket. 

The crystal structures of group II truncated hemoglobins from Bacillus subtilis (Bs-trHb1) [3], 

Thermobifida fusca (Tf-trHb) [4], Geobacillus stearothermophilus (Gs-trHb) [5] and Mycobacterium 

tuberculosis (Mt-trHbO) [6, 7] revealed a very polar heme pocket. In Bs-trHb and Gs-trHb, there is a 

H-bonding network involving Tyr B10, Gln E11 and the nitrogen atom of the tryptophan residue 

in position G8 whereas in Tf-trHb and Mt-trHbO and in the trHbO from P. haloplanktis TAC125 [8], 

an additional potential hydrogen bond donor is provided by a Tyr CD1 residue. Due to their very 

high oxygen affinity it is unlikely that these proteins may act as oxygen transporters, but their 

physiological role is not yet fully understood. The combination of electronic absorption, Resonance 

Raman, and Electron Paramagnetic Resonance spectroscopies is particularly useful in highlighting 

the structural properties of these proteins. In the present work the characterization of 

representative members of bacterial Hbs belonging to Group II together with selected site directed 

mutants of the amino acids of the distal side will be discussed. The following points will be 

treated: i) the strength of the proximal Fe-His bond via the ν(Fe-His) mode; ii) the steric effects 

imposed by the protein moiety on the structure of the heme macrocycle via the enhancement of the 

out-of-plane modes of the heme; iii) the interaction between the iron-bound ligand and the polar 

residues of the distal heme pocket. In particular, the vibrational frequencies of the Fe-Ligand 

complexes formed in the presence of carbon monoxide [9], fluoride, hydroxide, and hydrogen 

sulfide will be discussed in terms of specific interaction between the bound ligand and the distal 

amino acid residues. 

References 

[1] J. B.,Wittenberg, M. Bolognesi, B. A. Wittenberg, M. Guertin, J. Biol. Chem. 277, 871-874 (2002). 

[2] G. Wu, L. M. Wainwright, R. K. Poole, Adv. Microb. Physiol. 47, 255-310 (2003). 

[3] A. Giangiacomo, L. Ilari, A. Boffi, V. Morea, E. Chiancone, E. J. Biol. Chem. 280, 9192-9202 (2005). 

[4] A. Bonamore, A. Ilari, L. Giangiacomo, A. Bellelli, V. Morea, A. Boffi, FEBS J. 272, 4189-4201 (2005). 

[5] A. Ilari, P. Kjelgaard, C. von Wachenfeldt, B. Catacchio, E. Chiancone, A. Boffi, Arch. Biochem. Biophys. 457, 85- 

94 (2007). 

[6] M. Milani, A. Pesce, M. Nardini, H. Ouellet, Y. Ouellet, S. Dewilde, A. Bocedi, P. Ascenzi, M. Guertin, L. Moens, J. 

M. Friedman, J. B. Wittenberg, M. Bolognesi, M. J. Inorg. Biochem. 99, 97-109 (2005). 

[7] M. Milani, P. Y. Savard, H. Ouellet, P. Ascenzi, M. Guertin, M. Bolognesi, M. Proc. Natl. Acad. Sci. U.S.A. 100, 

5766-5771 (2003). 

[8] D. Giordano, D. Parrilli, A. Dettaï, R. Russo, G. Barbiero , G. Marino, G. Lecointre, G. di Prisco, L. Tutino, C. 

Verde, Gene 398, 69–77 (2007). 

[9] A. Feis, A. Lapini, B. Catacchio, S. Brogioni, P. Foggi, E. Chiancone, A. Boffi, G. Smulevich, Biochemistry 47, 

902-910 (2008). 

13


Atomic force spectroscopic investigation of 

biorecognition events 

SALVATORE CANNISTRARO 

Biophysics and Nanoscience Centre and CNISM, University of Tuscia, 

Largo dell’Università, Viterbo, I-01100, Italy 

Life is based on biomolecular interactions, and atomic force spectroscopy (AFS) is nowadays the 

most powerful and widely used technique for investigating forces, energies and dynamics of 

biomolecular complex, at the single molecule level [1]. AFS allows to probe complexes one at time, 

under near-physiological conditions, and without labeling procedures. Moreover, in AFS 

biomolecular interactions occur between bound molecules, and thus biorecognition events taking 

place at cell surfaces are well mimicked. However, to gain reliable information, the immobilization 

of proteins to tips and supports has to be carefully controlled. The influence of the immobilization 

strategy on the efficiency and strength of biomolecular interactions has been widely characterized 

[2], also by optimizing the biomolecular orientation on the basis of computational docking 

predicted molecular complex configurations [3]. Moreover, AFS can be successfully used both to 

investigate complexes having very different affinities and also to reveal competitive binding 

mechanisms, thus gaining deeper information about molecular interactions. Experimental details 

and potentialities of AFS will be described, together with some AFS applications to the study of 

biological complexes of different nature, ranging from transient to competitive or ternary 

complexes. 

References 

Fig. 1 – Schematic illustration of an AFS experiment to probe the 

selective interaction between the tumor suppressor p53 and the 

electron-transfer protein Azurin, which shows anti-cancer activity. 

[1] P. Robert, A.-M. Benoliel, A. Pierres, P. Bongrand, J. Mol. Recognit. 20, 432-447 (2007). 

[2] B. Bonanni, A. S. M. Kamruzzahan, A. R. Bizzarri, C. Rankl, H. J. Gruber, P. Hinterdorfer, S. Cannistraro, Biophys. 

J. 89, 2783-2791 (2005); B. Bonanni, A. R. Bizzarri, S. Cannistraro, J. Phys. Chem. B 110, 14574-14580 (2006); 

M. Taranta, A. R. Bizzarri, S. Cannistraro, J. Mol. Recognit. 21, 63-70 (2008). 

[3] A. R. Bizzarri, E. Brunori, B. Bonanni, S. Cannistraro, J. Mol. Recognit. 20, 122-131 (2007); V. De Grandis, A. R. 

Bizzarri, S. Cannistraro, J. Mol. Recognit. 20, 215-226 (2007); M. Taranta, A. R. Bizzarri, S. Cannistraro, J. Mol. 

Recognit. 22, 215-222 (2009). 

14


Theoretical search for molecular mechanisms of 

photostability of building blocks of life 

ANDRZEJ L. SOBOLEWSKI 

Institute of Physics, Polish Academy of Sciences, PL-02668 Warsaw, Poland 

The DNA and RNA bases absorb strongly in the 200 – 300 nm range, which renders living matter 

potentially vulnerable to the UV components of sunlight. Nevertheles, DNA is inherently highly 

photostable, that is, the quantum yield of photochemical reaction products is very low. 

Apparently, ultrafast photophysical excited-state deactivation-process depopulates the excited 

states where destructive reactions can take place. The unstructured absorption spectra of 

aminoacids and polypeptides, the generally very low quantum yield of fluorescence (with the 

exception of tryptophan containing proteins) indicate similar mechanisms of photostability exist in 

these species. This presentation addresses the calculation of reaction paths and the corresponding 

energy profiles in excited electronic states of selected DNA bases and their dimers as well as 

aminoacids and short peptide chains using ab initio methods. Adenine and guanine-cytosine base 

pair are considered as examples of the former systems, while tyrosine-(H2O)2 and Gly-Phe-Ala 

peptide represent the latter. For isolated adenine the calculations reveal the reaction pathway 

which leads to a conical intersection (CI) with the ground state via out-of-plane deformation of the 

hetero-aromatic ring in the lowest excited singlet state [1]. For hydrogen-bonded systems (GC 

dimer, Tyr-(H2O)2, and GlyPheAla) another mechanism of deactivation is identified. This involves 

electron-transfer followed by single-proton-transfer processes along existing hydrogen-bonds. 

These reaction paths lead to CIs with the electronic ground state and provide a pathway for 

ultrafast radiationless deactivation [2-4]. These findings suggest that CIs of the excited electronic 

states with the electronic ground state play a universal role in the photochemistry of molecular 

building blocks of life (amino acids, DNA bases, and their complexes). The conical intersections are 

the origin of the exceptional photostability of these compounds, which may have lead to their 

selection at the very beginning of the biological evolution [5]. 

References: 

[1] S. Perun, A.L. Sobolewski, W. Domcke, J. Am. Chem. Soc. 2005, 127, 6257 

[2] L. Sobolewski, W. Domcke, C. Hättig, Proc. Nat. Acad. Sci. 102 (2005) 17903 

[3] L. Sobolewski, D. Shmesh, W. Domcke, J. Phys. Chem. A, 113 (2009) 542 

[4] D. Shmesh, A.L. Sobolewski, W. Domcke, J. Am. Chem. Soc., 131 (2009) 1374 

[5] C. Sagan, J. Theor. Biol., 39 (1973) 195 

15


Water in narrow pores 

CHRISTOPH DELLAGO 

Faculty of Physics, University of Wien, Austria 

Water confined into the interior channels of narrow carbon nanotubes or transmembrane proteins 

forms collectively oriented molecular wires held together by tight hydrogen bonds. Here, I will 

explore the thermodynamic stability and dipolar orientation of such one-dimensional (1D) water 

chains from nanoscopic to macroscopic dimensions. A simple dipole lattice model accurately 

recovers key properties of 1D-confined water when compared to atomically detailed simulations. 

In a major reduction in computational complexity, the dipole model can be represented in terms of 

effective Coulombic charges, which allows us to study pores of macroscopic lengths in equilibrium 

with a water bath (or vapor). At ambient conditions, the water chains filling the tube are 

essentially continuous up to macroscopic dimensions. The properties of nanopore water can be 

probed experimentally with dielectric spectroscopy. Our computer simulations, carried out for a 

simplified water model, demonstrate that the dielectric response of 1d water chains follows Debye 

behavior. Exploiting that the time evolution of the total dipole moment of a 1D water chain is 

determined by the diffusive dynamics of essentially uncorrelated defects, we have derived simple 

formulas for the susceptibility and relaxation times as a function of chain length. These 

expressions, verified in extensive computer simulations, permit to extract fundamental molecular 

information such as the defect energy and their diffusion constant from dielectric relaxation 

spectra. The implications of the dipolar order of nanoconfined 1D water for long-range proton 

transport are also discussed. 

16


ORAL PRESENTATIONS 

AUGUST 29 th Saturday 

17


18


Recent studies of coherent vibrational motion in 

biomolecules 

PAUL M. CHAMPION 

Physics Department, Northeastern University, 

360 Huntington Avenue, Boston, MA 02115 USA 

Recent studies [1-3] have demonstrated how either static or transient distortions along specific 

normal modes of heme proteins serve to activate coherent resonance Raman intensities, otherwise 

be forbidden by symmetry. The distortion-induced enhancement mechanism [3] is most relevant 

for low-frequency modes, which are susceptible to distortion by environmental forces. The protein 

can evidently “tune” or distort the heme to perform a multitude of different tasks and these 

distortions can be monitored by measuring Raman-active vibrational coherences. The results 

demonstrate that vibrational coherence spectroscopy is a sensitive probe of thermally accessible 

and functionally relevant heme distortions, which serve as biochemical reaction coordinates. 

The figure shows the 

low-spin CN-bound ferric 

heme coherence spectra for 

myoglobin (Mb) and 

horseradish peroxidase 

(HRP). Here, the heme is in 

an identical electronic state 

for both proteins. However, 

the heme crystallographic 

structures, as analyzed by 

normal coordinate structural 

decomposition, show that 

HRP and Mb induce 

predominant heme saddling 

and doming distortions, 

respectively. The coherence 

spectra clearly reflect this 

difference. Additional studies 

of cytochrome c will be 

presented that demonstrate 

how excitation within an 

iron-ligand charge transfer 

band appears to activate the 

redox-active heme ruffling 

mode near 60cm -1. 

References 

Comparison of low frequency spectra of HRP-CN and Mb-CN 

60fs pulse,λ ex =433nm, open band detection, KPi buffer, pH=7 

0 500 1000 1500 2000 2500 3000 3500 4000 0 50 100 150 200 250 300 350 400 

0 500 1000 1500 2000 2500 3000 3500 4000 0 50 100 150 200 250 300 350 400 

time(fs) 

[1] Flaviu Gruia, Minoru Kubo, Xiong Ye, Dan Ionascu, Changyuan Lu, Robert K. Poole, Syun-Ru Yeh , and Paul M. 

Champion, J. Am. Chem. Soc. 130, 5231-5244 (2008). 

[2] Flaviu Gruia, Minoru Kubo, Xiong Ye and Paul M. Champion, Biophys. J. 94, 2252-2266 (2008). 

[3] Minoru Kubo, Flaviu Gruia, Abdelkrim Benabbas, William Montfort, Estelle Maes, and Paul M. Champion, J. Am. 

Chem. Soc. 130, 9800-9811 (2008). 

19 

HRP-CN 

Mb-CN 

23 

38 

69 

40 

65 

96 

100 

127 

141 

152 

176 

184 

209 

233 

223 

267 

252 

268 

wavenumber(cm -1 ) 

283 

342 

343 

371


Green fluorescent protein as a matrix for the study of 

ultrafast proton transfer in proteins 

MINAKO KONDO, ISMAEL A. HEISLER AND STEPHEN R. MEECH 

School of Chemistry, University of East Anglia, Norwich NR4 7TJ, UK 

The green fluorescent protein (GFP) is well known as a key technology in fluorescence imaging. 

The wild type protein is also known to exhibit a unique excited state proton transfer (ESPT) 

reaction. 1 This reaction has been characterized through structural studies and ultrafast vibrational 

spectroscopy as involving long range proton transport along a three step proton wire. 2 While such 

ESPT is very rare in biology proton transport itself has many important functions. Since many 

mutants of GFP can be prepared and structurally characterized it is possible that photoinitiation of 

proton transport provides a unique means of studying proton transport dynamics in proteins in 

real time. In this presentation two examples will be given, utilizing ultrafast fluorescence and time 

resolved vibrational spectroscopy. In one example we short circuit the proton wire in the wtGFP 

and replace it with a single acceptor very close to the proton donor. In this way we create a low 

barrier H-bond of the type much discussed in the context of enzyme catalysis. 3 It is shown that the 

resultant ESPT is extraordinarily fast, occurring on a sub 100 fs timescale (Figure 1). The second 

mutant localizes GFP almost exclusively in its neutral A state, allowing us to reinvestigate the 

origin of the previously noted 1 ultrafast component of the ESPT. This component has not been 

characterized in detail, and may result from multiple pathways in the wt GFP structure. 

References 

Fig. 1 – Ultrafast fluorescence up-conversion of S65T H148D GFP. 

The sub 100 fs dynamics in the blue region of the spectrum are 

assigned to ultrafast proton translocation on a low barrier H-bond. 

[1] M. Chattoraj, B.A. King, G.U. Bublitz, S.G. Boxer: PNAS 93 8362-67 (1996). 

[2] M. K. Brejc, T.K. Sixma, P.A. Kitts, S.R. Kain, R.Y. Tsien, M. Ormo, S.J. Remington: PNAS 94 2306-11 (1997); D. 

Stoner-Ma, A.A. Jaye, P. Matousek, M. Towrie, S.R. Meech, P.J. Tonge: J. Amer. Chem. Soc. 127 2864-65 (2005); 

J.J. van Thor, G. Zanetti, K.L. Ronayne, M. Towrie: J. of Phys. Chem. B 109 16099-108 (2005). 

[3] W.W. Cleland, M.M. Kreevoy: Science 264 1887-90 (1994). 

20


Time-Resolved diffusion and thermodynamics revealed 

spectrally hidden dynamics of phototropins 

YUSUKE NAKASONE 1 , SATORU TOKUTOMI 2 , MASAHIDE TERAZIMA 1 

1. Department of Chemistry, Graduate School of Science 

Kyoto University, Kyoto, Japan 

2. Department of Biological Science, Graduate School of Science, Osaka 

Prefecture University, Sakai, Osaka 599-8531, Japan 

Revealing molecular mechanism of a protein reaction has been a central issue not only in 

chemistry but also in more general science. For that purpose, a variety of time-resolved 

spectroscopic methods have been developed. However, they can monitor only dynamics 

associated with an optical transition and it has been very difficult to trace processes without 

optical transition. In this respect, one of powerful ways to characterize materials is diffusion 

and/or thermodynamics. However, a difficulty of the traditional techniques in studying chemical 

reactions is the lack of time-resolution. Recently our group has been working on time-resolved 

studies during a variety of photochemical processes in nano - milliseconds. We used the pulsed 

laser induced transient grating (TG) method for quantitative measurements in time domain. We 

applied this technique to the photochemical reactions of various proteins. Here we will show 

studies on phototropin. Phototropin is a blue light sensor protein of plants and the reaction 

dynamics has been attracting a lot of attention recently. From the TG signal, we could identify 

many intermediates, which have not been observed previously. It was found that the TG signals 

after photoexcitation of Arabidopsis phototropin 1 LOV2 (phot1LOV2) domain without the linker 

were very sensitive to the temperature; the diffusion signal increased drastically with increasing 

the temperature. The signal was consistently explained well in terms of superposition of the photoinduced 

dissociation and association reactions. This fact indicates presence of equilibrium between 

the monomer and the dimer of the phot1LOV2 domain in dark and this equilibrium was 

confirmed by a gel chromatographic technique. Interestingly, the TG signal of phot1LOV2 with the 

linker (phot1LOV2-linker), which exists as the monomer form was also temperature dependent; 

the diffusion signal intensity decreased with increasing the temperature. Since the diffusion signal 

reflected the conformation change of the linker upon the photoexcitation, this temperature 

dependence should indicate that there are two kinds of phot1LOV2-linker; one of them can exhibit 

a conformation change of the linker region upon the photoexcitation and the other does not. The 

fraction of these species depended on the temperature. Considering the monomer-dimer 

equilibrium of phot1LOV2 domain, we suggested that the non-reactive form possesses the linker 

region that is dissociated from the LOV2 domain. A 

relation with the biological function will be described. 

References 

Fig. 1 Schematic showing the equilibrium 

between the dimer and monomer of 

phot1LOV2 and between reactive and 

non-reactive states of phot1LOV2-linker 

sample. 

[1] Y.Nakasone, T.Eitoku, D.Matsuoka, S.Tokutomi, M.Terazima, J.Mol.Biol., 367, 432-442(2007).: Y. Nakasone, T. 

Eitoku, K. Zikihara, D. Matsuoka, S.Tokutomi, M. Terazima, J.Mol.Biol., 383, 904-913 (2008). 

21


Ultrafast protein response in BR and SRII: 

Results from isotopically labeled and chemically 

modified retinal proteins 

R. GROSS 1 , C. SCHUMANN 1# , N. FRIEDMAN 2 , M. SHEVES 2 , L. LI 3 , M. ENGELHARD 3 , 

O. TRENTMANN 4 , E. H. NEUHAUS 3 AND R. DILLER 1 

1. Dept. of Physics, University of Kaiserslautern, E. Schrödinger Str., D- 

67663 Kaiserslautern, Germany 

2. Dept. of Organ. Chemistry, Weizmann Institute of Science, 76100 

Rehovot, Israel 

3. Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Str. 11, D- 

44227 Dortmund 

4. Dept. of Biology, University of Kaiserslautern, E. Schrödinger Str., D- 

67663 Kaiserslautern, Germany 

# Current address: Leibniz Institute for New Materials, D-66123 

Saarbrücken, Germany 

The light driven function of retinal binding proteins in archaea depends critically on the coupling of 

the activated chromophore to the hosting protein moiety. In order to explore the dynamics of this 

process we have performed ultrafast transient mid-infrared spectroscopy on isotopically labeled 

samples of bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) [1]. The experiments allow the 

discrimination of chromophore [2,3] and non-chromophore bands and include SRII in D2O-buffer, 

BR in H2 18O medium, SRII with 15N-labeled protein and BR with 13C14 13C15-labeled retinal 

chromophore. Via observed shifts of infrared difference bands after photoexcitation and their 

kinetics we provide evidence for non-chromophore bands in the amide I and the amide II regions 

of BR and SRII. A band around 1550 cm -1 is very likely due to an amide II vibration. In the amide I 

region, contributions of modes involving exchangeable protons and modes not involving 

exchangeable protons can be discerned. Observed bands in the amide I region of BR are not due to 

bending vibrations of protein bound water molecules. The observed protein bands appear within 

0.3 – 3 ps and decay partially on a slower time scale of 9 – 18 ps, clearly showing that BR and SRII 

respond to chromophore activation on the time scale of the primary photoreaction. Our findings 

fill a gap concerning spectroscopic and time resolved evidence for the ultrafast coupling between 

the excited and isomerising chromophore and its protein environment. They are related to recent 

results on BR containing a sterically locked, non isomerizing chromophore (BR5.12), where similar 

protein bands have been observed [4]. Thus, the results not only shed light on the reaction 

mechanism in retinal proteins but also suggest implications for the coupling between an excited 

chromophore and a surrounding protein matrix in general. 

References 

[1] R. Gross, M. M. N. Wolf, C. Schumann, N. Friedman, M. Sheves, L. Li, M. Engelhard, O. Trentmann, H. E. Neuhaus, 

R. Diller, JACS, submitted (2009) 

[2] J. Herbst, K. Heyne and R. Diller, Science 297, 822-825 (2002) 

[3] R. Diller, R. Jakober, C. Schumann, F. Peters, J. P. Klare and M. Engelhard, Biopolymers, 82, 358–362 (2006) 

[4] R. Gross, C. Schumann, M.N.N. Wolf, J. Herbst, R. Diller, N. Friedman and M. Sheves, J. Phys. Chem. B. 113, 

7851–7860 (2009) 

22


Laser amplifier for spectroscopy of protonated peptides 

Oleg V. Boyarkin, Monia Guidi, Natalia S. Nagornova and Thomas R. Rizzo 

Laboratoire de Chimie Physique Moléculaire, École Polytechnique Fédérale de Lausanne, CH-1015 

Lausanne, Switzerland 

Specific biological functions of proteins are largely determined by their 3-D structures that they 

adopt in vivo. Calculation of these complex structures requires experimental verification and, 

perhaps, gradual adjustments of the employed theoretical models. This can be done by predicting 

structures of small species in an isolated environment, then extending to larger but still isolated 

peptides and proteins and finally to solvated species. Infrared (IR) spectroscopy of biomolecules 

in the gas-phase can provide such data in the form of sets of vibrational frequencies that is to be 

reproduced by a valid theory. Each set, if measured on the same conformer of a molecule, 

constitutes a spectroscopic signature that can be used to test and calibrate theory. We employ an 

IR-UV laser double resonance photo-fragmentation approach to measure conformer-selective IR 

spectra of protonated peptides, cooled to T=10K in a 22-pole linear ion trap.[1,2] The UV induced 

statistical photofragmentation slows quickly upon increasing size of peptides, limiting utility of 

this approach to small species. We report here a spectroscopic technique that allows an increase in 

the photofragmentation yield of large molecules of more than two orders of magnitude. In this 

approach protonated species that have been first excited electronically in the UV undergo infrared 

multiple photon excitation (IRMPE) by a pulsed TEA CO2 laser.[3] This additional vibrational 

excitation accelerates statistical dissociation rate and, therefore, increases fragmentation yield of 

the excited species on a time-scale of our experiment. We have verified that the CO2 laser assisted 

photofragmentation doesn’t distort UV spectra. It is also compatible with IR-UV depletion 

technique, allowing us to extend conformer specific IR spectroscopy to larger protonated peptides 

containing up to 17 amino acids and to a strongly bounded cyclic peptide - a natural antibiotic 

gramicidin-s.. 

References 

[1] O. V. Boyarkin, S. R. Mercier, A. Kamariotis, and T. R. Rizzo, J. Am. Chem. Soc. 128 (9), 2816 

(2006). 

[2] J. A. Stearns, S. Mercier, C. Seaiby, M. Guidi, O. V. Boyarkin, and T. R. Rizzo, J. Am. Chem. Soc. 

129 (38), 11814 (2007). 

[3] M. Guidi, U. J. Lorenz, G. Papadopoulos, O. V. Boyarkin, and T. R. Rizzo, The Journal of 

Physical Chemistry A 113 (5), 797 (2009). 

23


Spectroscopic and modeling studies of biomolecular 

adsorption on solid surfaces 

D. K. HORE, K. C. JENA, S. A. HALL AND T. TRUDEAU 

Dept. of Chemistry, University of Victoria, 

Victoria, British Columbia, V8W 3V6, Canada 

The interaction of biomolecules with solid surfaces is a fundamental aspect of many physiological, 

environmental, and technological processes. Often, the aim is to promote the specific adsorption 

of a particular protein to a surface. In a biosensor, for example, optimization is reliant on control 

of both the orientation and conformation of the protein to a substrate. While there are many 

excellent tools for probing bulk structures of biomolecules, there are relatively few techniques that 

can provide quantitative information on adsorbed structures. One of the challenges is that 

molecular features of the substrate and solution conditions are highly influential on the adsorbed 

biomolecular structure. Another is that a high degree of surface-specificity is required to 

discriminate from adjacent bulk phases. Our lab is focussed on developing spectroscopic and 

modeling techniques to probe such interactions and provide a feature-rich description of adsorbed 

biomolecular structure. Using techniques such as visible-infrared sum-frequency generation 

spectroscopy and molecular dynamics simulations, our aim is to arrive at the details of the 

submolecular interactions that govern the interplay between residue-residue and residue-surface 

interactions. 

References 

Fig. 1 – Three-wave mixing experiments performed with different 

polarizations of the input and output beams are revealing of 

orientation and conformation of molecules at the surface. The 

spectra are fit [1,2] to determine elements of the second-order 

susceptibility. This in turn is related to the hyperpolarizability in the 

molecular frame, thereby providing the link to surface-adsorbed 

structure [3]. 

[1] D. Hore, J. King, F. Moore, D. Alavi, M. Hamamoto, G. Richmond, Appl. Spectrosc. 58, 1377 (2004). 

[2] D. Hore, D. Beaman, G. Richmond, J. Chem. Phys. 121, 12589 (2004). 

[3] D. Hore, D. Beaman, D. Parks, G. Richmond, J. Phys. Chem. B 109, 16846 (2005). 

24


Structural analysis of macromolecular complexes 

by Isotope-Edited FTIR Spectroscopy 

Suren A. Tatulian 

Department of Physics, University of Central Florida, Orlando, FL, USA 

Structure determination of large macromolecular complexes, such as multidomain proteins or 

protein-membrane complexes, is one of the most challenging tasks in modern structural biology. 

High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of 

moderate size or those that can be crystallized easily, and protein-membrane systems are out of 

reach of both methods. This work describes an emerging biophysical technique that combines 

segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which 

provides detailed structural information on protein-membrane complexes and multidomain 

proteins. Labeling of a segment of the protein with 13C results in infrared spectral resolution of the 

labeled and unlabeled parts and thus allows identification of structural changes occurring in 

specific domains/segments of the protein that accompany functional changes. Segmental isotope 

labeling also allows determination of the precise configuration of protein-membrane complexes by 

polarized attenuated total reflection FTIR spectroscopy. These new developments offer solutions to 

functionally important site-specific structural changes in proteins and protein-membrane 

complexes that are hard to approach using conventional methods. 

25


Exciton dynamics and energy disorder in 

photosynthetic light-harvesting complexes 

V. Koning 1 , N. Verhart 1 , R. Purchase 1 , R. J. Silbey 1 and S. Völker 1,2 

1 Huygens and Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands. 2 Faculty of 

Sciences, Vrije Universiteit, Amsterdam, The Netherlands. 3 Department of Chemistry, 

Massachusetts Institute of Technology, Cambridge, MA, USA. 

In photosynthesis, solar energy is converted into chemical energy that drives subsequent metabolic 

reactions. The process begins with the absorption of photons by light-harvesting (LH) complexes 

from which the excitation energy is rapidly transferred to the reaction center (RC). In the latter, 

charge separation takes place and chemical energy becomes available for further processes. Purple 

bacteria have two types of LH-complexes, a core complex surrounding the RC called LH1, and a 

peripheral LH2 complex. The LH2 complexes are organized in two concentric rings: the B800 ring 

with well separated and weakly interacting bacteriochlophyll (BChl) molecules absorbing at ~ 800 

nm, and the B850 ring with strongly interacting BChls absorbing at ~ 850 nm. The strong 

interaction in B850 leads to delocalization of the electronic excitation. The degree of this 

delocalization, which is limited by static and dynamic disorder, remains a subject of debate and it 

is not clear whether the controversial results reported in the literature are related to the different 

techniques used and/or the differences in the bacteria studied. To clear up these controversies and 

to get a better understanding of the interplay between the coherence of the excitation originating 

from the strong electronic coupling and the energy disorder in the ring that tends to destroy the 

coherence, we have performed spectral hole-burning experiments detected in fluorescence on the 

B850 band of the LH2 complexes of four types of purple bacteria. By measuring the depth of the 

holes as a function of wavelength, we were able to determine the distribution of the lowest k=0 

exciton states within the B850 band. From the comparison of our experimental results with the 

simulations, we could get an estimate of the amount of energy disorder and electronic coupling in 

the various bacteria studied. Problems with the theoretical model and its implications will be 

discussed. 

26


Fluorescence and Infrared Cross-Correlation Spectroscopy: 

A new tool in analysing protein conformational coupling 

S. MADATHIL 1 , U. ALEXIEV 2 AND K. FAHMY1 1 

1. Div. of Biophysics, Institute of Radiochemistry, Forschungszentrum 

Dresden-Rossendorf, PF 510119, D-01314 Dresden, Germany 

2. Dept. of Physics, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany 

The functional regulation of proteins is based on structural changes that are initiated at a 

ligand-binding site and are transmitted to a "distant" site where biological activity is altered. 

Understanding the molecular mechanisms of long range coupling is crucial for many systems 

ranging from energy conversion and catalysis to pharmacoligical interference with receptors. 

We have developed a generalized multidimensional spectoscopic approach to investigate long 

range coupling in proteins by integrating simultaneously recorded fluorescence emission and 

infrared absorption from a protein sample undergoing conformational transitions in response 

to an external perturbation. Using fluorescence-coupled attenuated total relfectance (ATR) 

Fourier-transform infrared (FTIR) difference spectroscopy, coupling of the cytosolic 

conformation of the retinal protein rhodopsin to internal carboxyl_H-bonds has thus been 

identified [1]. Here we have used the fluorescence-labeled bacteriorhodopsin mutant D96A to 

show how the closure of the cytosolic half channel of this proton pump is coupled to the 

protonation state of the retinal Schiff base and its counter ion Asp85. Channel closure is 

strongly coupled to deprotonation of Asp85 but less to the reprotonation of the Schiff base 

which partially precedes the cytosolic conformational change. Azide accelerates channel 

closure relative to the internal protontransfer steps. In addition to site-specific labelling, the 

model-free analysis of cross-correlated spectral data can be extended to proteins containing 

natural flurophores. We show for the cytoskeletal protein actin that the loss of liganddependent 

static quenching of tryptophan emission during thermal unfolding correlates with 

the loss of specific secondary structure monitored by FTIR spectroscopy. This approach 

provides structural information on flavonoid binding to actin which has recently been shown 

to affect actin conformational changes [2]. Further topological information can be obtained 

from the emission wavelength of the tryptophans that become unquenched during ligand 

dissociation. Fluorescence-IR-cross-correlation spectroscopy thus extends the IR-based 

conformational analysis by site-specific information on local physical parameters (polarity, 

electrostatics, etc.) which affect the emission of intrinsic or extrinsic fluorophores. 

References 

[1] N. Lehmann, U. Alexiev, K. Fahmy, J. Mol. Biol. 336, 1129–1141 (2007). 

[2] M. Boehl, S. Tietze, A. Sokoll, S. Madathil, F. Pfennig, J. Apostolakis, K. Fahmy, H.-O. Gutzeit, 

Biophys. J. 93, 2767-2780 (2007). 

27


Shape of the carbon monoxide infrared absorption band 

of carboxyheme proteins as a probe of the protein 

anharmonicity. 

S. S. STAVROV 

Sackler Institute of Molecular Medicine, Dept. of Human Molecular 

Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv 

University, Ramat Aviv, Tel Aviv, 69978, Israel 

Heme proteins are intensively used to study chemistry and physics of proteins, because they can 

be studied by virtually any spectroscopic technique. Carboxyheme proteins manifest very strong 

infrared absorption band in the region of 1900 – 2000 cm -1, which corresponds to the valence 

vibration of the heme-coordinated CO molecule. Position of this band was shown to be affected by 

the protein environment [1, 2] and was used to study the HP structure and dynamics (see for 

review [2]). Modern infrared spectroscopy allows obtaining high resolution spectra of this band 

with high signal-noise ratio. Therefore not only position, but also shape of the band is used to 

obtain information about the protein structure and dynamics [3-5]. In this study we analyze 

general properties of shape of the CO band. It was shown earlier [6] that decay weakly contributes 

to the band width (~ 0.5 cm -1 at room temperature), whereas the band’s full width at half 

maximum is ~ 10 cm -1. Therefore, the main contribution to the CO band broadening stems from 

interaction of the CO vibration with motion of its environment, whereas the natural width of the 

band is negligibly small. If this environment moves harmonically, the band shape can be described 

in terms of theory of multi-phonon optical absorption band [6]. Using this theory we show that (a) 

the CO band is broadened by motions of the environment slower than ~10 ps (most probably they 

correspond to some large-amplitude motions of the protein); (2) the temperature dependence of 

the second moment of the band has to be linear in any temperature interval at T > 20 K. Deviation 

from the linearity points at anharmonic character of motion of the environment; (3) if at any 

temperature the spectrum is symmetric, it must be Gaussian. Experimental observation of another 

symmetric bandshape (for example, Voigtian) implies that motion of the environment is 

anharmonic, and the band is a superposition of Gaussians, each of which corresponds to a 

conformational substate of the protein. These results explain the experimentally observed 

temperature dependence of the CO band [5] and, in particular, bring one to a conclusion that even 

in dry trehalose protein conformational transition occurs. 

References 

[1] S. S. Stavrov, “The FeCO unit vibrations as a probe of the structure and dynamics of the active site of heme 

proteins: combined quantum chemical, vibronic and spectroscopic study”, in Biopolymer Research Trends, edited 

by R. B. Hamil, Nova Publishers, 119 (2008). 

[2] T. G. Spiro, I. H. Wasbotten, J. Inorg. Biochem. 99, 34-44 (2005) 

[3] J. D. Muller, B. H. McMahon, E. Y. T. Chien, S. G. Sligar, G. U. Nienhaus, Biophys. J. 77, 1036-1051 (1999) 

[4] A. Cupane, M. Leone, V. Militello, Biophys. Chem. 104, 335-344 (2003). 

[5] A. D.Kaposi, J. M. Vanderkooi, S. S. Stavrov, Biophys. J. 91, 4191-4200 (2006). 

[6] K. A. Merchant, D. E. Thompson, Q. H. Xu, R. B. Williams, R. F. Loring, M. D. Fayer, Biophys. J. 82, 3277-3288 

(2002). 

[7] Y. E. Perlin, Sov. Phys. Uspekhi 80, 553-595 (1964). 

28


Low-frequency dynamics of Bacteriorhodopsin studied 

by terahertz time-domain spectroscopy; relation with its 

function 

S. KAWAGUCHI 1 , M. SHIBATA 2 , H. KANDORI 2 , AND K. TOMINAGA 1,3 

1. Graduated School of Science, Kobe University 

2. Nagoya Institute of Technology, Showa-ku, Nagoya, 466-8555, Japan 

3. Molecular Photoscience Research Center, Kobe University, Nada-ku, 

Kobe, 657-8501, Japan 

When proteins express their functions, large conformational changes often occur. These changes 

result from collective motions of a large number of atoms. Such motions have characteristic 

frequencies in the region below a few tens of wavenumbers. Furthermore, it is well known that 

when proteins express their functions water molecules trapped internally and those surrounding 

the proteins play an important role. In this work, we have measured the low-frequency spectra of 

bacteriorhodopsin (BR) at various conditions of hydration and temperature using terahertz (THz) 

time-domain spectroscopy to discuss relation with expression of their function. From the obtained 

spectra of the refractive index and absorption coefficient we calculated Reduced Absorption Cross 

Section (RACS) in the THz region which is proportional to vibrational density of state (VDOS) [1]. 

It was found that the RACS of the BR samples shows a power-law behavior (RACS ν α ). At room 

temperature, the power-law of the dry sample is α = 1.97 ± 0.02.� The value of the exponent 

α �becomes smaller as the amount of hydration increases. By comparing the ideal case, anharmonic 

coupling among the low-frequency modes of BR becomes larger as the amount of hydration 

increases. Furthermore, the temperature dependence of the exponent is similar for both the dry 

and hydrated samples in the temperature range from -100 C to -40 C. However, above -40 C the 

hydrated samples show stronger temperature dependence than the dry samples. It shows that for 

the hydrated sample anharmonic coupling is induced above -40 C by increasing temperature. This 

change is due to the dynamical transition that was reported by the inelastic neutron scattering 

study [2]. 

References 

RACS (m 2 mol -1 ) 

10 

1 

0.1 

6 

4 

2 

6 

4 

2 

6 

5 6 7 8 9 

10 

Wavenumber (cm -1 ) 

29 

2 3 4 5 6 7 8 

Fig.1 Reduced Absorption Cross Section of BR in the THz region. 

[1] K. Yamamoto, et al. Biophys. J. 89, L22-L24 (2005). 

[2] M. Ferrand, et al. Biophys. J. 90, L9668-L9672 (1993).


Low temperature FTIR spectroscopy reveals new 

insights on the pH-dependent proton pathway of 

Proteorhodopsin 

G. SCHÄFER 1 , M.-K. VERHOEFEN 2 , S. SHASTRI 3 , 

I. WEBER 3 , C. GLAUBITZ 3 , J. WACHTVEITL 1,2 , W. MÄNTELE 1 

1. Institute of Biophysics, Max von Laue-Str.1, Johann Wolfgang Goethe-University Frankfurt , 60438 

Frankfurt, Germany 

2. Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University Frankfurt, Max von 

Laue-Str.7, 60438 Frankfurt, Germany 

3. Institute of Biophysical Chemistry and Center of Biomolecular Magnetic Resonance, Johann Wolfgang 

Goethe-University Frankfurt, Max von Laue-Str.9, 60438 Frankfurt, Germany 

Proteorhodopsin (PR) discovered in the γ-proteobacterium SAR86 is believed to be a light-driven 

proton pump. 1 Since the first description of PR several studies have uncovered evidence of 

intermediates comparable to the bacteriorhodopsin (BR) photocycle. However, photocycle and pH 

dependent proton transport of PR are still controversially discussed. Until now no M intermediate 

could be detected kinetically at acidic pH, but recently low temperature visible spectroscopy 

shows a band typical for the M state at pH 4. 2,3,4 We were able to characterize the main photocycle 

intermediates with a combined low-temperature infrared and visible spectroscopy approach. 

These thermodynamically controlled conditions allowed us to identify an M intermediate not only 

at pH 9.0 but also at pH 5.1. At both conditionstransport is enabled by the presence of a proton 

acceptor and concomitant changes in the protein structure. A tentative band assignment places 

Glu-108 as proton acceptor at low pH in contrast to its role as proton donor at pH 9.0. A 

bidirectional proton pumping above and below the pKa value of Asp-97 is thus likely. In addition, 

the photocycle properties are influenced by small pH changes in the order of 0.5 pH units. At 

pH 5.5 and pH 8.5 the difference spectra show distinctive differences connected to changes in the 

protonation state of key residues. We shed light on the complicated pH-dependence of the PR 

photocycle and were able to identify three distinct mechanisms connected to alkaline, neutral and 

acidic conditions leading to a holistically understanding of proton translocation in PR. 

References 

[1] Béjà, O. et al., Science, 289, 1902-1906 (2000). 

[2] Friedrich, T. et al., J. Mol. Biol., 321, 821-838 (2002). 

[3] Dioumaev, A.K. et al., Biochemistry, 42, 6582-6587 (2003). 

[4] Lörinczi, É., Verhoefen, M.-K. submitted. 

30


Time resolved EPR investigation on oxygen and 

temperature effects on a synthetic eumelanin 

F. CONTI 1 , L. PANZELLA 2 , A. NAPOLITANO 2 , M. D’ISCHIA 2 , AND A. TOFFOLETTI 1 

1. Dept. of Chemical Sciences, University of Padova, Via Marzolo 1, 

Padova, I-35131, Italy 

2. Dept. of Organic Chemistry and Biochemistry, University of Naples 

Federico II, Via Cintia 4, Naples, I-80126, Italy 

Eumelanins are the fundamental components of the mammalian pigmentary system. Their 

biological functions, in all the different localization, e.g. the skin or the retinal pigment epithelium, 

are influenced by uncommon physicochemical features [1]. Eumelanin properties include a 

permanent EPR signal due to a number of intrinsic quinone/semiquinone like radicals and a 

reversibly generated EPR signal under UV and visible irradiation, that suggests the formation of 

additional extrinsic radicals. Recently, TR-EPR studies have demonstrated that in the 

photoproduction of new melanin free radicals, they are generated as Radical Pairs from the triplet 

manifold [2]. Here, we compare photoreactivity of a synthetic eumelanins as function of the 

temperature in the range 290-140K. Moreover, we analyze the electron spin polarization of the EPR 

signal in the presence and in the absence of oxygen. TR-EPR spectra of samples under vacuum 

exhibit only a net emission after the exciting laser pulse, while for sample in equilibrium with 

oxygen in the air an additional distinct pattern consisting of an enhanced absorption followed by 

emission is present at the earliest times (delay


Oligonucleotide-based chemical nucleases: 

opportunities and challenges in RNA targeting 

STEVEN M. MILES 1 , MENGISTEAB B. GEBREZGIABHER 1 , WALEED A. ZALLOUM 1 , DMITRII V. 

PYSHNYI 2 , NADEZHDA L. MIRONOVA 2 , MARINA A. ZENKOVA 2 , VALENTIN V. VLASSOV 2 AND ELENA 

V. BICHENKOVA 1 

1. School of Pharmacy, University of Manchester, Oxford Road, 

Manchester, M13 9PT, U.K. 

2. Institute of Chemical Biology and Fundamental Medicine SB RAS, 

Novosibirsk, Russia 

The development of novel biomimetic supramolecular catalysts based on natural ribonucleases 

and capable of cleaving RNA targets presents an opportunity for new biological tools, or even 

therapeutics, affecting specific messenger RNAs and viral genomic RNAs. Our recent work on 

Peptidyl oligonucleotide-based Chemical Nucleases (PCN) 1-4 revealed a new type of chemical 

nuclease with unusual catalytic and structural properties. These novel deoxyoligonucleotide-based 

chemical nucleases were constructed by chemical conjugation of short catalytically inactive 

oligopeptides (containing alternating basic and hydrophobic amino acids) with 

deoxyoligonucleotide components, which are poorly or non-complementary to the RNA target 

region. After conjugation, the oligonucleotide component seemed to induce an active conformation 

for the peptide and hence significantly enhanced its catalytic performance. However, neither 

structural aspects of the active conformation(s) nor the fine mechanisms of cross-modulated 

conformational re-arrangement and subsequent cleavage were studied. It is our current aim to 

discover structure-activity relationships for the PCN systems using 1D and 2D NMR spectroscopic 

techniques ( 1H, 31P, COSY, NOESY) to inform the design of catalysts with enhanced properties. 

Here we report our first results on structural aspects of selected PCN systems using NMR 

spectroscopy in combination with computational studies. 

References 

[1] D. Pyshnyi et al. Nucleosides & Nucleotides, 16, 1571–1574 (1997). 

[2] N. Mironova et al. Nucl. Acids Res. 32, 1928-1936 (2004). 

[3] N. Mironova et al. J. Biomol. Struct. Dyn., 23, 591-602 (2006). 

[4] N. Mironova et al. Nucl. Acids Res. 35, 2356-2367 (2007). 

32


Molecular stress in biological membranes measured by 

solid-state NMR spectroscopy 

H. I. PETRACHE 

Department of Physics, Indiana University Purdue University 

Indianapolis, Indianapolis, IN 46202, USA 

The interfacial region between biological membranes and the aqueous space differs markedly from 

isotropic solutions [1]. Water molecules as well as interfacial solutes, although highly mobile and 

in permanent exchange with the bath, are dynamically restricted (ordered) on membrane surfaces 

[2]. In addition, the interactions within biological membranes which govern the function of ion 

channels and membrane receptors are also highly anisotropic. How to measure and describe these 

interactions that govern the function of biological membranes? Solid state NMR methods are well 

suited to provide experimental observables to address such questions. In particular, 2H and 31P 

NMR provides valuable information on the molecular dynamics at the membrane-water interface 

[2], while 2H NMR of deuterated lipid probes provide information on the molecular interactions 

within membranes [3]. We report and analyze experimental data to provide a description of 

molecular interactions in terms of a mean-torque profile obtained from solid-state 2H NMR. Two 

aspects will be addressed: the role of charged lipid headgroups on interfacial interactions and the 

role of polyunsaturated lipid chains on the membrane forces. We show that lateral compression 

energies generated by such molecular substitutions are sufficiently large to provide a 

thermodynamic driving force for protein conformational changes. This approach provides a 

predictive framework for relating lipid composition to membrane function. 

References 

[1] H. I. Petrache et al., PNAS 103, 7982 (2006) 

[2] H. I. Petrache et al., Biophys. J. 86, 1574 (2004) 

[3] K. Rajamoorthi et al., J. Am. Chem. Soc. 127, 1576 (2005) 

33


Self-assembled ion channels 

probed by nitroxide spin labels and PELDOR spectroscopy 

A. D. Milov 1 , R. I. Samoilova 1 , Y. D. Tsvetkov 1 , F. Formaggio 2 , 

C. Toniolo 2 , J.-W. Handgraaf 3 and J. Raap 4 

1. Institute of Chemical Kinetics and Combustion, Novosibirsk, 630090 Russian Federation 

2. Institute of Biomolecular Chemistry, CNR, Department of Chemistry, University of Padova, 

35131 Padova, Italy 

3. Culgi B.V., P.O. Box 557, 2300 AN Leiden, The Netherlands 

4. Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 

2300 RA Leiden, The Netherlands 

Alamethicin is a membrane active peptide antibiotic which contains 19 amino acid residues and a 

phenylalaninol residue at the C-terminus. Interest in this molecule is provoked by its ability to change 

the permeability of biological membranes by forming voltage-gated conductive channels. In contrast to 

protein channels, peptide induced channels are generally believed to be formed by self-association of a 

number of amphipathic helical molecules. The mechanism of opening and closing the channel might be 

triggered by this self-assembling process. From the concentration dependences of the conductances it is 

known that 2-11 alamethicin molecules might be involved in the transport of small metal cations 

through the membrane barrier. Pulsed electron-electron double resonance (PELDOR) was used to 

study the structure of aggregates of mono spin labelled alamethicin molecules in the membranes of 

large multilamellar phosphocholine vesicles, which were 

frozen to 77K. This method, combined with the application 

of nitroxide spin labels which are rigidly connected to well 

defined positions of the peptide backbone allowed us to 

determine the aggregate number and distances between 

spin labels in the range of 1.5-8 nm as well. In the 

presentation the quaternary structure of the 

supramolecular cluster of molecules will be presented in 

terms of a penknife structural model. 

Fig.1 Energy minimized model of the supramolecular 

alamethicin tetramer wherein the spatial positions of the 

spin labels are indicated by balls, (A) A side view of the 

tube model shows the arrangement of four parallelly 

aligned α-helical peptide chains. The maximum diameter of the peptide complex (3.2 nm) appears 

roughly matching the hydrophobic thickness of the membrane double layer (3.5 nm). The electric 

dipole-dipole interactions between the polar side chains (inset) are believed to stabilize the C-terminal 

ends of the peptide chains. (B) Top down view of the peptide backbone orientations. (C) A semitransparent 

surface model of a thin slice (thickness of about half of a helix-turn) showing the entry of 

one or more channels within the tetramer. 

Reference A. D. Milov, R. I. Samoilava, Yu. D. Tsvetkov, F. Formaggio, C. Toniolo and J. Raap, J. Am. 

Chem. Soc. 129, 9260-9261 (2007) 

34



AUGUST 30 th Sunday 

35


36


Tracking structural changes in solution with 100 ps 

time-resolved X-ray scattering 

MARCO CAMMARATA 

ESRF, Grenoble, France 

Reactions are at the core of life and chemistry. The fast time scales usually involved have 

prevented direct structural studies up to very recently. In the last years the intrinsicly pulsed 

nature of synchrotron sources has been used to allow laser pump / x-ray probe studies with a time 

resolution of 100ps. 

Examples coming from chemistry and biophysics will be discussed together with the key 

ingredients of the experimental technique. 

X-ray free electron lasers will allow to push the time resolution even further but wavelength and 

position stability, together with other beam characteristics, will be crucial for the success of such 

experiments. 

References 

[1] Cammarata, M. et al., Nature Methods, 5, 881 (2008) 

[2] Ihee, H. et al., Science, 309, 1223 (2005) 

[3] Cammarata, M et al., Rev. Sci. Instrum. 80: 015101 (2009) 

37


Proteins in action monitored by time-resolved FTIR spectroscopy 

KLAUS GERWERT 

Ruhr-Universität Bochum, Lehrstuhl für Biophysik, Bochum 

In the Postgenom era one of the remaining major challenges is the detailed understanding of protein networks within the 

living cell. Currently, there is a large gap between the detailed understanding of proteins in vitro and their description in 

interaction pathway maps in systems biology. In order to contribute to a more detailed understanding of protein 

interactions a combined approach of x-ray structure analysis, time-resolved FTIR spectroscopy, Molecular Dynamic and 

QM/MM biomolecular simulations is used. Time-resolved FTIR difference spectroscopy can be used to monitor the 

reactions within proteins at atomic resolution with ns time-resolution up to days [1]. This provides in combination with 

structural models in addition spatial resolution. Complementary, by QM/MM simulations theoretical IR spectra can be 

obtained. More quantitative information is thereby deduced from the IR spectra. Based on fast scan studies on 

bacteriorhodopsin the key catalytic residues, asp 85 and asp 96 and their protonation kinetics are identified and 

summarized in a first detailed proton pump model [2]. Based on succeeding step scan FTIR measurements the interplay 

between protein bound water molecules, a strongly hydrogen bonded water, a dangling water and a protonated water 

complex is elucidated in detail. It results in a controlled Grotthus proton transfer from the central proton binding site to 

the protein surface [3]. A similar mechanism might apply in the photosynthetic reaction center [4]. Using caged GTP the 

GTPase mechanism of the protooncogen Ras is investigated [5]. The ras protein switches external signals to the nucleus. 

It is down regulated by a protein-protein interaction with the GAP protein which catalyses the GTP hydrolysis by five 

orders of magnitude. Oncogenic mutations in Ras prevent this catalysis, which results in uncontrolled cell growth. The 

Ras-GAP protein interaction is be studied time-resolved [6,7]. This provides a detailed thermodynamic characterisation 

of the catalytic mechanism. It is shown that the movement of a catalytic GAP-“arg-finger” into the GTP binding site, 

pushes water molecules out of the binding pocket. Thereby the activation entropy is increased and the hydrolysis is 

catalysed [8,9]. The studies proves that the trFTIR approach can be applied to protein-protein interactions. Beside 

reaction within the active site of a protein, also the surface change of a protein, which controls the protein-protein 

interactions is monitored [10]. In order to investigate the protein interactions closer to physiological conditions the ATR 

(attenuated total reflection) technique is applied. Using this approach for the first time the folding of the prion protein 

bound to a rafts lipid bylayer is studied [11]. 

References 

(1) Kötting, C., Gerwert, K., Chem Phys. Chem 6, 881-888 (2005) 

(2) Gerwert, K., Hess, B., Soppa, J., Oesterhelt, D., Proc. Natl. Acad. Sci USA 86, 4943-4947 (1989) 

(3) Garczarek, F., Gerwert, K. Nature 439, 109-112 (2006) 

(4) Remy, A., Gerwert, K., Nature Struct. Biol. 10, 637-644 (2003) 

(5) Cepus,V., Goody, R.S., Gerwert, K., Biochemistry 1998, 37, 10263-10271, (1998) 

(6) Allin, C., Ahmadian, M. R., Wittinghofer, A., Gerwert, K., Proc. Natl. Acad. Sci., USA 98, 7754 (2001) 

(7) Kötting, C., Blessenohl, M., Suveyzdis, Y., Goody, R.S., Wittinghofer, A., Gerwert, K. Proc. Natl. Acad. Sci. USA 

103, 13911-13916 (2006) 

(8) te Heesen, H; Gerwert, K.; Schlitter, J., Febs Letters, 581, 5677-5684 (2007) 

(9) Kötting, C., Kallenbach A., Suveyzdis, Y., Wittinghofer, A. Gerwert, K., Proc. Natl. Sci., 105, 17, 6260-6265 (2008) 

(10) Kötting, C., Kallenbach, A., Suveyzdis, Y., Eichholz, C., Gerwert, K., ChemBioChem, 8, 781-787 (2007) 

(11) Elfrink, K., Ollesch, J., Stöhr, C., Willbold, D., Riesner, D., Gerwert, K.., Proc. Natl. Acad. Sci, 105, 31, 10815- 

10819 (2008) 

38


Dynamic crossovers and quantum effects 

in protein hydration water 

F. Bruni (1), S. E. Pagnotta (2) 

(1) Dipartimento di Fisica, Universit_a degli Studi di Roma Tre, Rome, Italy 

(2) Centro de Fisica de Materiales (CSIC-UPV/EHU) - Materials Physics Center MPC, 

Donostia-San Sebastian, Spain 

Recent work has focused on dielectric relaxation experiments on hydrated proteins, aimed 

at the study of water and protein dynamics [1- 4]. At variance with these experiments, we 

will report on the dielectric relaxation of water protons. In particular, we discuss the 

temperature dependence of proton dynamics along chains of hydrogen bonded water 

molecules at the interface with a globular protein. The rationale behind this approach is 

that measurements of the proton mobility are closely linked to the dynamics and 

connectivity of the water molecules in the protein hydration shell. 

Quantum effects on the water proton dynamics over the surface of a hydrated protein are 

measured by means of broadband dielectric spectroscopy and deep inelastic neutron 

scattering [5] 

[1] S. Pawlus, S. Khodadadi, and A. P. Sokolov, Phys. Rev. Lett. 100, 108103 (2008). 

[2] S. Khodadadi, S. Pawlus, J. H. Roh, V. Garcia-Sakai, E. Mamontov, and A. P. Sokolov, J. Chem. Phys. 128, 

195106 (2008). 

[3] S. Khodadadi, S. Pawlus, and A. P. Sokolov, J. Phys. Chem. B. 112, 14273 (2008). 

[4] H. Frauenfelder, G. Chen, J. Berendzen, P. W. Fenimore, H. Janssonb, B. H. McMahon, I. R. Stroe, J. 

Swenson and Robert D. Young, Proc. Nat. Acad. Sci. 106, 5129 (2009). 

[5] S. E. Pagnotta, F. Bruni. R. Senesi, and A. Pietropaolo, Biophys. J. 96, 1939 (2009) 

39


Folding dynamics of peptides studied by temperaturejump 

infrared-spectroscopy 

C. KREJTSCHI 1 , O. RIDDERBUSCH 1 , R. HUANG 2 , L. WU 2 , T. A. KEIDERLING 2 , K. HAUSER 1 

1. Institut für Biophysik, Universität Frankfurt, Max-von-Laue-Str. 1, 

60438 Frankfurt, Germany 

2. Department of Chemistry, University of Illinois at Chicago, 845 W. 

Taylor St. Chicago, Illinois 60607- 7061, USA 

Peptides with well-defined secondary structure are ideal model systems to study protein folding 

mechanisms. Infrared techniques provide both the necessary time resolution as well as the 

structural sensitivity. The amide I’ region, mainly consisting of the coupled C=O stretching 

vibrations of the polypeptide backbone, is a sensitive marker for secondary structure and 

structural changes. We initiate rapid heating by laser-excited ns temperature jumps (~10°C) and 

study fast ns-to-µs relaxation dynamics [1]. The dynamics of the alpha-helix to random coil 

transition of polyglutamic acid was analyzed under reversible folding/refolding pH-conditions. 

The observed relaxation kinetics allowed separation of the folding and unfolding process with 

additional use of FTIR measurements in thermal equilibrium. Site-specific dynamics have been 

monitored for a set of isotopically labeled beta-hairpin peptides, variants of a 12-mer tryptophan 

zipper whose conformation is stabilized by a hydrophobic core formed from the interaction of four 

tryptophan residues. Various single and cross-strand coupled 13C=O labeled variants have been 

analyzed by probing the relaxation kinetics via separate partially resolved 13C=O amide I’ bands. 

Differences in the kinetic behavior have been found for the loss of beta-strand and the gain of 

disordered structure. The isotope-edited kinetics show variations in local structural stability of the 

hairpin backbone. Our data support a multistate dynamic behavior that prevents clear 

determination of folding and unfolding time constants. Nonetheless, the site-specific kinetics are 

consistent with a hydrophobic collapse hypothesis for hairpin folding [2]. The contribution of the 

hydrophobic core to the hairpin stability has additionally been analyzed with mutants of this 

sequence whose tryptophan residues have been selectively substituted by valines. 

References 

[1] C. Krejtschi, R. Huang, T.A. Keiderling, K. Hauser, Vibr. Spec. 48, 1-7 (2008) 

[2] K. Hauser, C. Krejtschi, R. Huang, L. Wu, T.A. Keiderling, J. Am. Chem. Soc. 130, 2984-2992 (2008) 

40


Photoemission and the shape of free amino acids 

V. FEYER 1 , O. PLEKAN 1 , R. RICHTER 1 , M. CORENO 2,3 , M. DE SIMONE 3 , K.C. PRINCE 1,3 , 

W. ZHANG 4,5,6 , V. CARRAVETTA 6 , 

1. Sincrotrone Trieste, Basovizza (Trieste), I-34012, Italy. 

2. Istituto di Metodologie Inorganiche e dei Plasmi, Montelibretti (Rome), I-00016, Italy. 

3. Laboratorio Nazionale TASC, Basovizza (Trieste), I-34012, Italy. 

4. Department of Theoretical Chemistry, School of Biotechnology, Royal 

Institute of Technology, Stockholm, S-10691, Sweden. 

5. Hefei National Laboratory for Physical Science at the Microscale, 

University of Science and Technology, Hefei, Anhui 230026, P.R. China. 

6. Institute of Chemical Physical Processes, Pisa, I-56124, Italy. 

Low energy spectroscopies such as microwave and infrared techniques are commonly used as methods to 

determine the structure of free biomolecules, but high energy techniques like x-ray photoemission 

spectroscopy (XPS) have not yet yielded much significant structural information about biomolecules in the 

gas phase. Using XPS we have measured eight amino acids (Gly, Ala, Thr, Met, Pro, Trp, Phe and Tyr) at 

the C, N and O 1s edges. These amino acids all contain a single nitrogen atom in an amino group (NH2), 

except Trp which has an additional nitrogen atom in the indole ring. The N 1s core level spectra of Pro, Ala, 

Thr, Pro, Phe and Tyr show an additional shoulder or two peaks (Fig. 1a) [1-3] instead of the single peak 

expected from the stoichiometry. Gly and Met show single narrow N 1s peaks [3]. High quality calculations 

demonstrate that the peaks are due to different conformers, OH•••N strong hydrogen bonds and NH2•••O=C 

weak hydrogen bonds (Fig. 1b). We have also shown using XPS we can measure some populations of 

conformers at the measurement temperature experimentally, which is extremely difficult otherwise. In 

addition XPS has an advantage that the temperature of the sample is well known, and therefore some 

thermodynamic parameters (entropy, enthalpy) can be determined [1, 3]. 

Intensity (arb. units) 

References 

a) 

SUM 

PHE-1 

PHE-2 

PHE-3 

PHE-6 

407.0 406.0 405.0 404.0 

Binding energy, eV 

Fig. 1 a) N 1s photoemission spectra of phenylalanine, points: experimental data; 

thick solid lines: calculated spectra; thin solid lines: fits. b) Structures of the four 

lowest energy conformers of phenylalanine [4]. 

[1] O. Plekan, V. Feyer, R. Richter, M. Coreno, M. de Simone, K.C. Prince, V. Carravetta, Chem. Phys. Lett. 442, 429-433 (2007). 

[2] V. Feyer, O. Plekan, R. Richter, M. Coreno, K.C. Prince, V. Carravetta, J. Phys. Chem. A. 112, 7806-7815 (2008). 

[3] O. Plekan, V. Feyer, R. Richter, M. Coreno, M. de Simone, K.C. Prince, V. Carravetta, J. Phys. Chem. A, 111, 10998-11005 (2007). 

[4] W. Zhang, V. Carravetta, O. Plekan, V. Feyer, R. Richter, M. Coreno, K.C. Prince, J. Chem. Phys. submitted (2009). 

41


42


A new method to monitor ligand binding to proteins, 

amide I band simulations and the infrared spectrum of 

acetyl phosphate 

S. KUMAR, N. EREMINA, E.-L. KARJALAINEN, H. KUMAR, M. RUDBECK, A. BARTH 

Dept. of Biochemistry and Biophysics, Stockholm University, Arrhenius 

Laboratories, 10691 Stockholm, Sweden, barth@dbb.su.se 

Results from several projects will be presented: (i) Ligand binding to four different proteins was 

detected with infrared spectroscopy via an absorbance change of the OH stretching band of water. 

The effect is likely caused by a transfer of hydration water into bulk water. In principle, this 

method can be applied to any molecular association event and may therefore become an important 

new technology for drug development. (ii) The amide I band of beta sheet assemblies was 

calculated according to ref. [1]. The calculations reveal that the infrared spectrum is sensitive to the 

assembly of two sheets into a beta sheet stack. When the distance between the two sheets is 7 Å, an 

upshift of up to 5 cm -1 is observed relative to two isolated sheets, which depends on the relative 

orientation of the two sheets. This is relevant for the Alzheimer's peptide which forms a stacked 

beta sheet. Our calculations of the amide I band have been further refined by including 

information from density functional theory calculations [2] as well as the effects of hydrogen 

bonding and test calculations will be presented. (iii) In our attempt to understand how Ca 2+ 

pumping by the Ca 2+-ATPase is controlled by the environment of the phosphoenzyme phosphate 

group, we have studied the vibrational spectrum and the bond lengths of the model compound 

acetyl phosphate in simple environments constituted by HF molecules. HF bonding to the 

bridging oxygen and to the phosphorus atom has the largest effects on the above properties in line 

with our experimental spectra [3] and with interactions observed in crystal structures. 

References 

[1] H. Torii, M. Tasumi, J. Chem. Phys. 96, 3379-3387 (1992). 

[2] J.-H. Choi, M. Cho, J. Chem. Phys.120, 4383-4392 (2004). 

[3] A. Barth, N. Bezlyepkina, J. Biol. Chem. 279, 51888-51896 (2004). 

43


The regulation of the formation of cytoskeletal protein 

complexes by actin-binding proteins 

T. KUPI, Z. UJFALUSI, SZ. BARKÓ, J. BELÁGYI, G. HILD AND M. NYITRAI 

Dept. of Biophysics, University of Pécs, Szigeti str. 12., Pécs, H-7624, 

Hungary 

In living cells various groups of proteins are associated to supramolecular actin filament 

structures. The nature of the actin-binding proteins forming complexes with actin depends on the 

actin nucleation factors which initiated the polymerisation of the filaments. For example, actin 

structures associated with formins can bind tropomyosin and profilin, while those polymerised by 

the nucleation of the Arp2/3 complex bind cofilin and myosin I. Although these observations are 

known for a long time, the molecular mechanisms underlying the regulation of the formation of 

these protein complexes is still ambiguous. By using various biophysical methods we have shown 

recently that one of the actin nucleation factor families, the formins, can bind the actin filaments 

and change their conformational state. The formin-bound filaments are more flexible than those 

formed in the absence of formins [1, 2, 3]. We have also shown that subsequent binding of other 

actin-binding proteins, such as tropomyosin [4] and myosin, can reverse these changes. It appears 

that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined 

and specific binding site, and thus it is able to stabilise a certain conformation of the actin 

polymers. The altered conformational state of the actin filaments observed after the binding of 

these proteins, especially the formins, provides a possible explanation for the modified affinity of 

the filaments for other-actin binding proteins. We assume that the affinities are modified 

differently by different nucleation factors. As actin nucleation factors are the first protein to 

interact with the filaments, the conformational changes introduced to actin by these actin 

nucleation factors can serve as the molecular bases for the regulation of the formation of actin 

based intracellular protein complexes. Experiments are in progress to test and further corroborate 

the existence of such regulatory mechanisms in living cells.l. 

References 

[1] B. Bugyi, G. Papp, G. Hild, D. Lırinczy, E.M. Nevalainen, P. Lappalainen, B. Somogyi and M. Nyitrai. J. Biol. 

Chem., 281(16), 10727-36 (2006). 

[2] G., Papp, B. Bugyi, Z. Ujfalusi, Sz. Barkó, G. Hild, B. Somogyi and M. Nyitrai. Biophys. J., 91(7), 2564-2572 

(2006). 

[3] T. Kupi, P. Gróf, M. Nyitrai and J. Belágyi. Biophys. J., 96(7), 2901-2911 (2009). 

[4] Z. Ujfalusi, A. Vig, G. Hild and M. Nyitrai. 2009. Biophys. J., in press (2009). 

44


Study of dynamic properties of reconstituted myelin 

sheath. 

W. KNOLL 1 , F. NATALI 2 , J. PETERS 1 AND P. KURSULA 3 

1. University Joseph Fourier and Institut Laue-Langevin, Grenoble, FR- 

38000, France 

2. CNR-INFM, OGG, c/o Institut Laue-Langevin, Grenoble, FR-38000, 

France 

3. University of Oulu, Oulu, Finland 

Myelin is the lipid-rich, multilamellar membrane discontinuously wrapped around nerve axons, 

which increases the efficiency of saltatory impulse conduction (up to 350 km/hr in a healthy 

person). The molecular components of the myelin sheath interact tightly with each other and 

molecules on the axonal surface to drive myelination, to keep both myelin and the axon intact, and 

to transduce signals from myelin to the axon and vice versa. Myelin is destroyed by autoimmune 

processes in human demyelinating diseases, including multiple sclerosis (MS) in CNS and 

peripheral neuropathies (PN). Despite the presence of a well-defined set of myelin-specific 

proteins, little is known about the dynamics of these proteins and their influence on myelin 

stability. We present here first neutron scattering results on the dynamics of the myelin sheath and 

of the interaction between its constituents. In particular, we will try to clarify the influence of the 

myelin proteins on the myelin stability. 

45


Disulfide chromophore and its optical activity 

L. BEDNÁROVÁ 1 , P. MALOŇ 1 , H. DLOUHÁ 1 , M. KUBÁŇOVÁ 2 AND V. BAUMRUK 2 

1. Institute of Organic Chemistry and Biochemistry, Flemingovo nám. 2, 

Prague 6, 166 10, Czech Republic 

2. Charles University in Prague, Faculty of Mathematics and Physics, 

Institute of Physics, Ke Karlovu 5, Prague 2, 121 16, Czech Republic 

The use of various spectroscopic methods and their chiroptical variants, for the 

determination of peptide/protein conformation is relatively well established [1, 2]. 

Although the information is of rather low resolution it can be obtained for samples in 

solution and therefore has a distinct advantage over more informative methods like NMR 

or X-ray crystallography. Electronic circular dichroism (ECD) measured in the visible and 

near UV spectral region carries majority of structural information via the amide group. 

Detailed analyses of ECD give also additional structural data about other functional 

groups existing in peptide/protein molecules. These involve aromatic chromophores of 

Phe, Tyr and Trp side chains, the not very well understood contribution of the imidazole 

ring of histidine and a contribution of cystine disulphide chromophore, which is 

sometimes detectable as the high wavelength tail of the CD spectrum. Disulphide group is 

the only chromophore in proteins and peptides, which by itself exhibits inherent chirality 

and therefore should give rise to substantial chiroptical manifestation in electronic spectra 

(the non-planar disulphide chromophore itself is of C2 symmetry). In practice, it is 

unfortunately not the case and especially the low energy CD bands of the disulphide 

group with the maximum at about 260 nm are low in intensity and rather broad. If we 

consider, in addition, the possible overlap with CD bands of aromatic chromophores of 

phenylalanine, tyrosine and tryptophan residues, it is not surprising that structure 

oriented application of electronic CD spectroscopy to a disulphide chromophore is quite 

difficult. In this contribution we scrutinize chiral disulphides by other variants of 

chiroptical spectroscopy, namely vibrational optical activity measured in Raman 

scattering [3]. Raman spectroscopy is for this purpose rather promising already in its non 

chiral variant (it gives information on the C-S bond conformation), but one should 

underline that the obtained information is not complete. In that way no information about 

‘absolute conformation’ of the disulphide bridge can be acquired. According to theoretical 

calculations [4] Raman optical activity could provide this very specific information using 

the S-S (~500 cm -1 ) and C-S (~700 cm -1 ) stretching vibrations. The ECD, IR and Raman 

spectra, VCD and ROA spectra of model systems are presented with the aim to cast light 

on this unresolved problem. The spectra are compared with theoretical predictions. 

References 

[1] R. W. Woody, A. K. Dunker, in Circular Dichroism: Conformational Analysis of Biopolymers, edited by G. D. 

Fasman, New York, Plenum Press, 109 (1996). 

[2] Spectroscopic Methods for Determining Protein Structure in Solution, edited by H. A. Havel, VCH Publishers, New 

York (1996). 

[3] J. Kapitán, V. Baumruk, H. Hulačová, P. Maloň, Vib. Spectrosc. 42, 88-92 (2006). 

[4] L. Bednárová, P. Bouř, P. Maloň, Chirality (submitted). 

46


Ultrafast Processes in Photosynthesis 

RIENK VAN GRONDELLE 

Faculty of Science, VU University, De Boelelaan 1081, 1081 HV, 

Amsterdam, The Netherlands 

Photosynthesis is the process by which plants, algae and photosynthetic bacteria store the energy 

of the sun in chemical free energy. The photosynthetic apparatus consists of a multitude of 

membrane-associated pigment-proteins, that display a high degree of supramolecular organization 

to optimally perform their function. Three ultrafast processes underlie the high efficiency of 

natural photosynthesis: (1) transfer of excitation energy among the pigments of the lightharvesting 

antenna, (2) transmembrane charge separation in the photosynthetic reaction center 

and (3) quenching of excitation energy under high-light conditions. In this talk I will illustrate how 

femtosecond laser spectroscopy has contributed to the unraveling of the molecular basis of these 

elementary events. 

47


Steady State and Time-Resolved Fluorescence 

Measurements of the Green Fluorescent Variants T203V 

and T203V/S205V 

RINAT GEPSHTEIN 1 , DAN HUPPERT 1 , XIAOKUN SHU 2 AND S. JAMES REMINGTON 2 

1. Raymond and Beverly Sackler Faculty of Exact Sciences, School of 

Chemistry Tel-Aviv University, Tel-Aviv, Israel. Huppert@tulip.tau.ac.il 

2. Institute of Molecular Biology and Department of Physics, University 

of Oregon, Eugene, OR 97403-1229 

Steady state emission and picosecond time-correlated single photon counting (TCSPC) 

measurements were used to study the excited state proton transfer reaction in the green 

fluorescent proteins T203V, T203V/S205V, S205T, S205G and S205A mutants in H2O and D2O. The 

mutation T203V blocks a possible escape route of the transferred proton from the barrel structure 

to the exterior. The dual mutation T203V/S205V blocks both the main proton pathway to the 

proton acceptor E222 and the escape route from the barrel thus disabling the excited state proton 

transfer and causing this mutant to emit only in the blue and lack the strong green fluorescence of 

the deprotonated form. We found that at room temperature the proton transfer rate of T203V is 

some what smaller than that of wt-GFP. We found that the kinetic isotope effect (KIE) of the 

proton transfer rate is about 5. We studied the temperature dependence of the ESPT process of 

T203V in H2O (pH 9) in the temperature range of 88-320K. The proton transfer rate constant 

exhibits a non Arrhenius behavior. In D2O at sufficiently low temperatures, T


The influence of activator on conformational changes in 

human platelet Integrin αIIbβ3 

A. MCNALLY 1 , R. STEFFAN 1 , C. DE CHAUMONT 2, N. MORAN 2, R.J. FORSTER 1 , T.E. KEYES 1 

1. School of Chemical Sciences, National Centre for Sensor Research, 

Dublin City University, Glasnevin, Dublin 9, Ireland. 

2. The Department of Clinical Pharmacology, Royal College of Surgeons 

in Ireland, Dublin 2, Ireland. 

Integrin αIIbβ3 is a heterodimeric receptor protein incorporated into the plasma membrane of 

human platelets. It plays a key role in linking platelets to extracellular adhesion molecules such as 

fibrinogen and the intracellular actin cytoskeleton and is crucial in haemostasis and thrombosis. It 

consists of two transmembrane subunits αIIb and β3 and the dimer possesses a large extracellular 

and short cytoplasmic tail domains which are non–covalently linked. The integrin exists in at least 

two conformational forms. In its native state, it assumes a resting configuration characterized by a 

low binding affinity to its primary ligand fibrinogen. In intact platelets the ligand binding function 

is activated by physiologic stimuli such as thrombin, collagen or ADP via an inside–out signaling 

mechanism, and integrin assumes an activated state with high affinity for fibrinogen binding.[1] A 

number of chemical activators are commonly employed in biophysical studies of purified αIIbβ3 

which are generally expected to mimic in vivo activation of integrin, including DTT (dithioreitol), 

Mn(II) and EDTA. However, to our knowledge no comparative study has been made on the 

structural consequences of activation with these reagents. In this contribution, we describe a 

systematic study of the effects of these reagents and a cyclic RGD heptapeptide antagonist of 

αIIbβ3, eptifibatide, on αIIbβ3 structure using Raman and circular dichroism spectroscopy and 

fluorescence lifetime studies. These studies are complimented by whole platelet studies using 

flow cytometry.[2] Our data suggest that these commonly employed activators yield active-like 

integrin states but that each reagent induces significantly different conformational change in the 

integrin. Activation also has a profound impact on the disulfide bonding in this cysteine rich 

protein. Raman spectroscopy reveals that in the native state, the disulfides show a high degree of 

steric strain whereby a substantial fraction of the CCSSCC rotamers existing in non-equilibrium 

geometry.[3] Activation reduces this strain, but the nature and extent of this change varies with 

activator. 

References 

[1] T. Xiao T, J. Takagi J-h. Wang, B.S. Coller T.A. Springer, Nature, (2004), 432, 59-67 

[2] G. M. Walsh, D. Leane, N. Moran, T.E. Keyes, R.J. Forster, D. Kenny, S O'Neill. Biochemistry, (2007) 46, 6429-36. 

[3] H.E. Van Wart, A. Lewis, HA. Scheraga, F.D and Saeva, PNAS, (1973) 70, 2619. 

49


50



AUGUST 31 st Monday 

51


52


Unfolded and (Self)-Aggregated States of Alanine 

Based Peptides 

R. SCHWEITZER-STENNER, A. HAGARMAN AND T. J. MEASEY 

Department of Chemistry, University of Palermo, 3141 Chestnut Street, 

Philadelphia, PA 19026, USA 

The unfolded state of peptides has attracted substantial interest owing to the growing awareness 

of the fact that it is structurally less random than expected [1]. Investigations have focused on 

alanine, owing to its abundance in all types of proteins. MD simulations generally support the 

statistical coil picture in that they predict nearly equal populations of helical and non-helical 

extended conformations [2]. Experimental data are thus far somewhat inconclusive. A substantial 

number of experiments clearly suggest that alanine prefers a polyproline II (PPII) like 

conformations which cluster around φ=-70 o and ψ=150 o, but the reported propensity scales differ 

substantially [1]. We used the structural sensitivity of excitonic coupling between amide I 

backbone modes to explore the structural manifold of various polyalanines by analyzing this 

mode’s band profiles in the respective IR, isotropic Raman, anisotropic Raman and vibrational 

circular dichroism specta. Additionally, we utilized the 3JNHCαH constants obtained from 1H NMR 

as input. The analysis is based on distribution models describing the sampling of the 

Ramachandran space. We found that alanine has indeed a high propensity for PPII, exhibiting an 

intrinsic propensity of ca. 0.78 (on a scale from 0 to 1). This is demonstrated by the distribution 

function for alanine in GAG (Fig. 1). This propensity is slightly increased (0.85), if A is flanked by 

other alanines [3]. Longer polyalanines are generally doped with ionized residues to guarantee 

their solubility. These peptides have been used for studying helix↔coil transitions. While alanine 

maintains its high propensity for PPII, preliminary data suggest that the charged residues are more 

conformational random thus giving rise to a somewhat more compact structure of unfolded 

polyalanines. To our surprise we found that not all doped polyalanines with more than 12 residues 

form a helix in aqueous solution. The 16-mer (AAKA)16 instantaneously self-aggregates into large 

scale β-sheets which subsequently decay into a disordered state with the usual high PPII content. 

References 

Fig. 1 – Conformational distribution of alanine in GAG. 

[1] Z. Shi, K. Shen, Z. Liu, N.R. Kallenbach, Chem. Rev. 106- (2007). 

[2] Y. Duan, C. Wu, S. Chowdury, M.C. Lee, G. Xiong, W. Zhang, R. Yang, P. Cieplak, R. Luo, T. Lee, J. Caldwell, J. 

Wang, P. Kollman, J. Comp. Chem. 24, 1999-2012, 2002. 

[3] R. Schweitzer-Stenner, J. Phys. Chem. B. in press, published as a.s.a.p. article, (2009). 

53


A novel approach to multiscale measurements in 

biological materials: silk as a model system 

C. DICKO 1 , A. RODGER 2 , S. V. HOFFMANN 3 , F. VOLLRATH 1 

1. Dept. of Zoology, University of Oxford, OX1 3PS, Oxford, UK 

2. Dept. of Chemistry, University of Warwick, CV4 7AL, Coventry UK 

3. Institute for Storage Ring Facility (ISA), University of Aarhus, DK8000, Aarhus C, Denmark 

In a recent and first application using Synchrotron Radiation Linear Dichroism (SRLD) we found 

that it was possible to characterize the UV spectra of a bundle of silk fibers. Furthermore, we were 

able to monitor the effect of fibers extension using the LD spectra. A direct plot of the reduced LD r 

(LD normalized by the isotropic absorption) in figure 1 (left) shows how silk transition dipoles 

lined respectively to the fiber axis, and how they behave under extension. Negative LD r 

corresponds to a molecular orientation perpendicular to the fiber axis. From a simple 

interpretation of the amide chromophores electronic transitions we could separate the spectra in 

figure 1 in regions corresponding to parts or domains of the silk protein. Direct plot of the LD r as a 

function of strain (data not shown) showed segmental mobility of the chains. If the silk were a 

uniform material one would predict a linear dependence of the LD r with strain and identical slopes 

at all wavelengths. Instead, we observed sequential mobility that can be linked to silk remarkable 

mechanical properties. Testing further the method we compared two silks of similar function but 

from closely related spiders (figure1-right) and found that their spectral signature and chains 

orientation varied dramatically. In the presentation we will explore the observed segmental 

mobility of silks in light of their measured mechanical properties and electronic/vibrational 

couplings. We will, finally, explore the role of SRLD and routine LD into resolving the structurefunction 

relationship in biological materials. 

Fig. 1 – Reduced LD spectra of spider major ampullate silk as function of wavelength and tensile 

deformation. Region (1) protein backbone, (2) side chains, (3), (4) others. Right panel, comparison of 

reduced LD signals of dragline silk from Nephila edulis and Argiope lobata spiders. 

54


UV-Vis and FT-IR spectra of ultraviolet irradiated collagen in the 

presence of antioxidant ascorbic acid 

N. Metreveli 1 , K. Jariashvili 2 , L. Namicheishvili 2 , E. Chikvaidze 2 , Alina Sionkowska 3 , J. 

Skopinska 3 

1. Faculty of Physics and Mathematics, Ilia. Chavchavadze State 

University, Chavchavadze Ave. 32, 0179 Tbilisi, Georgia 

2. Dept of Exact and Natural Sciences, Iv. Javakhishvili Tbilisi State 


3. Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87- 

100 Torun, Poland 

The influence of deleterious UV irradiation on collagen molecule in the absence and presence of 

ascorbic acid using UV-Vis and FT-IR spectroscopy has been studied. Intensity of UV-Vis 

absorption spectrum of collagen with a maximum at 275 nm due to the aromatic residues (tyrosine 

and phenylalanine) increases with the increasing dose of UV irradiation. This effect is significantly 

hindered in the presence of antioxidant ascorbic acid. Intensities of FT-IR bands (amide A, B, I and 

II) of collagen decrease with the increase of the UV irradiation dosage. Intensities of bands are also 

decreased in the presence of ascorbic acid. Obtained results suggest that collagen photo-stability 

increases, increasing the concentration of ascorbic acid and collagen becomes less sensitive toward 

the UV irradiation. Formation of hydrogen bonds between the groups N-H of collagen and C=O of 

ascorbic acid possibly takes place. Though more hypothetical is that under UV irradiation free 

radicals appearing in acid soluble collagen and evoking photo-degradation of macromolecule 

restore due to the ability of ascorbic acid donating one or two electrons [1,2]. Increasing the dose of 

irradiation more molecules of ascorbic acid are delayed and antioxidant effect is diminished 

accordingly. 

References 

[1] N. Metreveli, L. Namicheishvili, K. Jariashvili, G. Mrevlishvili, A. Sionkowska, International Journal of Photoenergy, 

Article ID 76830, 1-4 (2006). 

[2] N. Metreveli, L. Namicheishvili, K. Jariashvili, M. Dgebuadze, E. Chikvaidze, A. Sionkowska, Journal of 

Photochemistry and Photobiology B: biology, 93(2), 61-65 (2008). 

55


Equilibrium spectroscopic studies of aromatic-aromatic 

interaction and pH effect on trpzip β-hairpin stability 

Ling Wu 1 , Takahiro Takekiyo 2 , Dan McElheny 1 , Anjan Roy 1 , Timothy A. Keiderling 1 

1 Department of Chemistry, University of Illinois at Chicago, 

845 W. Taylor St., Chicago IL, 60607-7061 USA 

2 National Defense Academy, Hashirimizu Yokosuka, Kanagawa, Japan 

Aromatic side chains in proteins are often involved in pairs or larger networks, most of which 

form interacting networks of three or more aromatic side chains. Analysis of neighboring aromatic 

groups can lead to improved understanding of protein folding mechanisms and stability. We have 

used the β-hairpin forming peptide Trpzip2 [1] as a template to study the effect of aromaticaromatic 

interaction on peptide stability. Optical spectra (ECD, FTIR) were measured and NMR 

structures determined for the original TZ2 peptide [2] and its Tyr and Val-substituted mutants to 

characterize their conformation and thermal stability. The NMR structures showed the strongly 

interacting Trp-Trp edge-to-face geometry to be maintained in hairpins with just two Trps placed 

in directly cross-strand positions. Diagonal Trp-Trp interactions did not result in a stable hairpin 

fold. The structures for the Tyr-Tyr interactions show a different geometry, but still yield relatively 

constrained Tyr side chains. The thermal unfolding processes for three Val mutants were studied 

under both neutral and acidic conditions by IR and ECD. From analysis of the IR amide I’ band, 

which reflects peptide secondary structure, we see more random coil content at acidic pH than 

those at neutral pH. The transition temperatures obtained using both IR and CD are lower at acidic 

pH than at neutral pH, which suggests that this peptide is less stable at acidic pH. Thermodynamic 

analyses of the temperature dependent CD and IR spectra obtained at neutral pH showed two 

transition temperatures, indicative of a complex folding pathway. At lower pH, these become 

competitive, so that CD and IR analyses yield similar thermodynamic parameters. Tyr-Tyr shows 

much weaker aromatic-aromatic interaction than Trp-Trp, but that is stronger than just 

hydrophobic interactions as determined by comparison to Val substituting for Trp residues. 

Aromatic interaction shows a stronger effect in stabilizing this peptide. The CD spectra are not a 

simple sum of the CD for the cross-strand pairs of Trp, and we have shown by TD-DFT 

calculations that the diagonal interactions contribute to the CD if not much to the stability. Added 

studies of Trpzip1 based hairpins with Tyr substitutions gave consistent results.[3] 

References 

[1] Cochran et al, Proc.Nat.Acad.Sci.(USA), 98, 5578-5583 (2001) 

[2] Rong Huang, Ling Wu, Dan McElheny, Petr Bour, Anjan Roy, Timothy A. Keiderling, J. Phys. Chem. B 113, 5661-5674 (2009) 

[3] Takahiro Takekiyo, Ling Wu, Yukihiro Yoshimura, Akio Shimizu, and Timothy A. Keiderling, Biochemistry 48, 1543-1552 (2009) 

56


Denaturation of proteins with beta-barrel topology 

induced by guanidine hydrochloride 

OLESYA V. STEPANENKO 1 , I. M. KUZNETSOVA 1 , V. V. VERKHUSHA 2 , M. STAIANO 3 , S. D’AURIA 3 

AND K. K. TUROVEROV 1 

1. Institute of Cytology of the Russian Academy of Sciences, Tikhoretsky 

av. 4, 194064, Saint-Petersburg, Russia 

2. Albert Einstein College of Medicine, 1300 Morris Park Avenue, 10461, 

New York, USA 

3. Institute of Protein Biochemistry CNR, Via Pietro Castellino 111, 

Naples, 80131, Italy 

The investigation of protein folding is one of the most actual and rapidly rising trends of molecular biology. 

Folding of proteins with high content of β-structure and, especially, proteins with β-barrel topology is 

significantly less understood with respect to folding of α-helical proteins. This work is devoted to study of 

denaturation processes induced by guanidine hydrochloride (GdnHCl) of proteins possessing β-barrel 

structure: porcine odorant-binding protein (OBP) and a series of fluorescent proteins (FPs) and their mutants. 

A great interest to fluorescent proteins is caused by their wide use as optical reporters of numerous cell 

events. The odorant-binding proteins represent an exceptional interest because of investigation of olfaction 

mechanism and possibility to exploit these proteins as sensing probe of biosensors to different substances, 

including dangerous. Our results have revealed that proteins with β-barrel topology have a higher structural 

stability with regard to globular α-helical proteins. At the same time, the rate of OBP denaturation is higher 

in comparison to FPs, which indicates that the value of energy barrier between native and unfolded state for 

FPs exceeds that for OBP essentially. It should be pointed out that denaturant addition to the green 

fluorescent protein EGFP leads to the sharp increase of chromophore fluorescence intensity. Quasiequilibrium 

curves also have shown that the addition of 0.1-0.2 M GdnHCl to EGFP results in approximately 

20% increase of “green” fluorescence intensity. It have been concluded that in the presence of small GdnHCl 

concentrations the structure of EGFP becomes less strained. As a result, the chromophore being inaccessible 

to quenching action of water molecules gains more planar configuration. This causes the increase of πelectron 

system conjugation and, in its turn, the increase of EGFP fluorescence quantum yield. In the case of 

OBP, the pre-denaturating GdnHCl concentrations lead to the increase of tryptophan fluorescence quantum 

yield and the decrease of fluorescence lifetime of the single tryptophan residue of OBP, Trp16. These effects 

are not accompanied by noticeable changes of protein structure which is manifested by invariance of 

parameter A, fluorescence anisotropy, the contribution of tyrosine residues to the bulk protein fluorescence, 

and far-UV CD spectra. The accessibility of Trp16 to the solvent remains unchanged and near-UV CD 

spectra become more pronounced. These effects have been explained by local changes in microenvironment 

of Trp16 which leads to the destruction of Trp16 complex with the positively charged atom NZ of Lys 120, 

localized near Trp16 indole ring and to the formation of exciplex between Trp16 and bound water molecules 

in its close vicinity. Thus the small GdnHCl concentrations have been shown to induce local structural 

disturbances in proteins. Similar structural changes have been previously observed for a number of proteins: 

creatine kinase, actin, carbonic anhydrase. This work was supported by NATO (CLG.983088), Contract with 

FASI (02.512.11.2277) and Program "Leading Scientific School of Russia" (1961.2008.4). 

57


Determination of conformational changes during 

aggregation of the amyloid-beta peptide by attenuated 

total reflection-Fourier Transform Infrared 

Spectroscopy. 

R. SARROUKH 1 , E. CERF 1 , A. ITKIN 1 , S. DERCLAYE 2 , Y. DUFRÊNE 2 , J.-M. RUYSSCHAERT 1 , 

E.GOORMAGHTIGH 1 AND V. RAUSSENS 1 

1. Center for Structural Biology and Bioinformatics, Laboratory for 

Structure and Function of Biological Membranes, Faculté des Sciences, 

Université Libre de Bruxelles, CP 206/2, Blvd. duTriomphe, B-1050 

Brussels, Belgium. 

2. Unité de Chimie des Interfaces, Université Catholique de Louvain, 

Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium. 

Alzheimer’s disease is the most common form of dementia worldwide. One hallmark of this brain 

disorder is characterized by polymorphous extracellular deposits called "senile plaques". Amyloid- 

beta peptide (Aβ) is the primary component of these plaques and plays an important, but not 

completely understood, role in the neurotoxicity. Two major forms of Aβ are produced by the 

proteolytic cleavage of the amyloid precursor protein: 40 and 42 residue-long. The most abundant 

form is the 40 amino acids peptide. Aβ can form different entities: monomeric, large soluble 

entities collectively called oligomers and insoluble fibrils. In the early stages of aggregation, Aβ 

forms oligomers, which are now considered to be the most toxic forms. Continued aggregation 

gives rise to fibrils formation. The mechanisms leading to accumulation of misfolded peptide and 

the fibrils formation still remain a matter of debate. Studies report that Aβ becomes toxic for 

neurons upon aggregation [1]. We have previously shown, in our laboratory, that the oligomeric 

species and fibrils of Aβ42 display distinct spectral features by attenuated total reflection-Fourier 

transform infrared (ATR-FTIR) spectroscopy. Whereas fibrils display spectral features of parallel 

β-sheet, oligomers adopt an antiparallel β-sheet structure [2]. Based on these observations, we 

followed the aggregation of Aβ40 using ATR-FTIR. The aggregation was also followed by 

fluorescence spectroscopy using a probe (Thioflavine T, ThT) specific to fibrils. We observed that 

Aβ40 polymerized, in our case, first into antiparallel β-sheet oligomers which are recognized by a 

conformational antibody specific to these species (A11). Aβ moves from an antiparallel to a parallel 

β-sheet structure. This shift from oligomers to fibrils observed by ATR-FTIR was in perfect 

agreement with the marked increase in ThT fluorescence upon fibrils formation. This 

conformational change supposes a reorganization of the peptide upon aggregation, which is also 

observed using monoclonal antibodies region-specific used to study the accessibility of aggregates. 

We demonstrated that ATR-FTIR is a powerful tool for protein aggregation studies: ATR-FTIR (i) 

can discriminate between Aβ oligomers and fibrils from a structural point of view (ii) shows a 

conformational change in the structure of the peptide upon aggregation, perfectly correlated to 

the formation of fibrils, as probed by fluorescence. 

References 

[1] Selkoe DJ. Trends Cell. Biol.(1998) 8, 447-453 

[2] Cerf E, Sarroukh R, Tamamizu-Kato S, Breydo L, Derclaye S, Dufrêne Y, Narayanaswami V, Goormaghtigh E, 

Ruysschaert JM, Raussens V. Biochem J (2009) Accepted Manuscript. 

58


A 2DCOS study of the effect of radiation on TGase activity 

I.DELAARADA 1 , J.L.R.ARRONDO 1 J.GONZALEZ VELASCO 2 AND P.BILBAO 2,3 

1. Unidad De Biofísica (Csic-Upv) And Departamento De Bioquímica, 

Universidad del País Vasco, Bilbao, Spain. 

2. Depto. Cirugia, Radiología y Medicina Física , Universidad del País 

Vasco, Bilbao, Spain. 

3. Servicio de Oncología Radioterapeútica, Hospital de Cruces- 

Osakidetza, Bilbao 

Transglutaminases (TGase) are ubiquous enzymes activated only after major disruptions in 

physiological or homeostatic processes. However, their activity is strongly regulated since 

overexpression of the enzyme does not change cell survival. In mammalian cells 6 isozymes have 

been isolated even if the genome there are 8 different isozymes. The most characteristic is Factor 

<strong>XIII</strong>, involved in blood clotting activation. TGase-2 has been isolated from keratinocytes in soluble 

form or associated to membranes. The enzyme can be activated by physical agents such as 

ultraviolet or ionizing irradiation or by chemical agents such as the chemotherapeutic drugs. The 

changes produced in TGase activity under radiation have been attributed to metabolomic effects 

associated with variations in cell calcium levels. We have studies the changes in the presence of 

excess calcium and they are still present, pointing to a structural effect. In order to see the changes 

we have used TGase2 from hepatic cells being irradiated at different doses and measuring the 

amide I infrared spectrum at different times. As expected, since the cells irradiated are viable, the 

changes observed analyzing the decomposition are minimal, so we have used 2DCOS to analyze 

and visualize the changes. This approach shows that the changes observed in the amide I are 

consistent and allow us to propose that thee changes in activity produced in TGase upon radiation 

are due to structural changes rather to metabolomic alterations. 

Acknowledgement: This work has been supported by a grant (2006111064) from the Department of 

Health of the Basque Government 

59


Biomedical application of Mössbauer spectroscopy with 

high velocity resolution: revealing of small variations 

M.I. OSHTRAKH 1, V.A. SEMIONKIN 1,2 , O.B. MILDER 1,3 , I.V. ALENKINA 1,2 AND E.G. NOVIKOV 1,2 

1. Faculty of Physical Techniques and Devices for Quality Control, 

Physical–Technical Dept., Ural State Technical University – UPI, 

Ekaterinburg, 620002, Russian Federation 

2. Faculty of Experimental Physics, Physical–Technical Dept., Ural State 

Technical University – UPI, Ekaterinburg, 620002, Russian Federation 

3. Radio–Technical Dept., Ural State Technical University – UPI, 

Ekaterinburg, 620002, Russian Federation 

Mössbauer spectroscopy is a useful technique for study iron-containing biological species that was 

applied in biomedical research [1, 2]. Comparative study of small variations of the iron electronic 

structure and stereochemistry in proteins and model compounds is one of the interesting aims in 

this field [3]. This interest is related to protein heterogeneity due to natural evolution process or 

pathological changes related to molecular diseases and to the iron electronic structure sensitivity to 

protein molecular structure variations. However, it takes to improve velocity resolution in 

Mössbauer spectroscopy to detect small variations of the iron electronic structure well. Usually 

Mössbauer spectra are measured in 512 channels or less. We used high stable, precision and 

sensitive spectrometer SM-2201 with spectra measurement in 4096 channels. An increase of 

velocity resolution leads to decrease of the experimental error for hyperfine parameters and to 

more reliable fitting of complicated spectra. Therefore, we demonstrated comparison of the study 

of various biological subjects such as hemoglobins, ferritin and its pharmaceutically important 

models, and liver and spleen tissues using Mössbauer spectroscopy with low and high velocity 

resolution (see Fig. 1). These results demonstrated better revealing of small variations of 

Mössbauer hyperfine parameters permitted us to distinguish various proteins of the same type as 

well as proteins in normal and pathological case. This may be a good base for further development 

of diagnostic tests on the basis of Mössbauer hyperfine parameters. This work was supported in 

part by the Russian Foundation for Basic Research (grant # 09-02-00055-a). 

QUADRUPOLE SPLITTING, mm/s 

2.125 

2.115 

2.105 

2.095 

2.085 

2.075 

2.065 

0.245 0.255 0.265 0.275 0.285 

ISOMER SHIFT, mm/s 


0.750 

0.740 

0.730 

0.720 

0.710 

0.700 

0.690 

0.680 

0.670 

0.340 0.350 0.360 0.370 0.380 


60 

0.595 

0.585 

0.575 

0.565 

0.555 

0.545 

0.535 

a b c 


0.525 

0.370 0.380 0.390 0.400 0.410 


Fig. 1 – Small differences of Mössbauer hyperfine parameters: a – oxyhemoglobins: human 

(�, 512 ch.) and rabbit (�, 512 ch., �, 1024 ch.); b – human ferritin (�), Imferon (�) and 

Maltofer® (�), open symbols – 2048 ch., black symbols – 512 ch.; c – chicken spleen: � – 

normal and � – leukemia, open symbols – 1024 ch., black symbols – 512 ch. 

References 

[1] M. I. Oshtrakh, Hyperfine Interact. 165, 313–320 (2005). 

[2] M. I. Oshtrakh, J. Radioanal. Nucl. Chem. 269, 407–415 (2006). 

[3] M. I. Oshtrakh, Spectrochim. Acta, Part A: Molec. and Biomolec. Spectroscopy 60, 217–234 (2004).


Raman markers of cancer. 

Ultrafast dynamics of carotenoids and lipids 

Jakub Surmacki 1, Piotr Ciacka 1, Beata Brozek-Pluska 1, Joanna Jablonska 2, Radzislaw Kordek 2, 

Halina Abramczyk 1,3 

1 Technical University of Lodz, Chemistry Department, Laboratory of Laser Molecular Spectroscopy, 93-590 

Lodz Wroblewskiego 15 str Poland, e-mail abramczy@mitr.p.lodz.pl, 

2 Department of Oncology, Medical University of Lodz, Paderewskiego 4, 93-509, Poland 

3* Max-Born-Institute Fax: +49 30 6392 1409 Max-Born-Str. 2A 12489 Berlin Germany, e-mail: 

abramczy@mbi-berlin.de 

Our recent papers [1-3] on medical applications demonstrate power of Raman spectroscopy as a 

diagnostic tool for breast cancer diagnosis. The results demonstrate the ability of Raman 

spectroscopy to accurately characterize breast cancer tissue and provide evidence that carotenoids 

and lipids of the tissue play an essential role as a Raman biomarkers that are able to distinguish 

between normal, malignant and benign types. Fig. 1 shows a typical spectrum of normal and 

malignant breast tissues and their histopathological images. 

Fig. 1 – Raman spectra (non-dyed samples) and histopathological 

images of typical normal and malignant (infiltrating ductal cancer) 

breast tissues of the same patient performed at identical 

experimental conditions 

The role of carotenoids is disscused in terms of their ability to act as light harvesting antenna that 

protects tissue from oxidative damage, lipid-phase anti-oxidant and upregulator of junctional 

communication in connexins. The possible mechanisms of interaction between carotenoids and 

lipids are discussed. The role of water on energy dissipation rate at the water/lipid interface will 

be discussed. Femtosecond infrared two-color and bleaching pump-probe experiments were used 

to investigate vibrational relaxation dynamics of C-H stretch modes in the lipid alkyl chains of 2,3- 

Dipalmitoyl-sn-glycero-1-phosphocholine (DPPC) and O-H stretch mode of water at the 

water/lipid interface for a controlled humidity of the sample. 

References 

intensity (cts/s) 

6000 

5000 

4000 

3000 

2000 

1000 

0 

malignant tissue 

normal tissue 

1000 2000 3000 4000 5000 6000 7000 8000 

wavenumber (cm -1 ) 

[1] H. Abramczyk, I. Placek, B. BroŜek – Płuska, K. Kurczewski, Z. Morawiec, M. Tazbir, J. Mol. Liquid 141,145-148 (2008) 

[2] H. Abramczyk, I. Placek , B. BroŜek – Płuska, K. Kurczewski, Z. Morawiec, M. Tazbir, Spectroscopy 22, 113-121 (2008) 

[3] H. Abramczyk, J. Surmacki, B. BroŜek – Płuska, Z. Morawiec, M. Tazbir, The Hallmarks of Breast Cancer by Raman Spectroscopy, J. Mol. 

Struc. DOI: 10.1016/jmolstruc. 2008.10.055 

61


Far-IR synchrotron and low-wavenumber Raman 

studies of proteins and protein/water interactions. From 

model system to animal and human skin 

O.F. NIELSEN 1 , T.M. GREVE 1,2 , N. DE FRIES 1 , N.W. LARSEN 1 , K.B. ANDERSEN 2 ,A. ENGDAHL 3 

AND B. NELANDER 3 

1. Dept. of Chemistry, University of Copenhagen, Universitetsparken 5, 

Copenhagen, DK-2100, Denmark 

2. Dept. of Spectroscopy and Physical Chemistry, LEO-Phama, 

Industriparken 55, Ballerup, DK-2750, Denmark 

3. MAX-lab, Lund University, Ole Römers väg 1, Lund, SE-22363, 

Sweden 

The low-wavenumber part of the vibrational spectrum yields information about intermolecular 

hydrogen bonded interactions. Peptides, proteins, water/protein mixtures and samples of human 

and animal skin were investigated. Synchrotron radiation was used as a light source to record the 

FT-Far-IR spectra. The spectra were obtained through a microscope in either ATR- or transmission 

modes. Near-IR-FT-Raman spectra were recorded with excitation at 1064 nm. The spectra were 

transformed to the R( ν) -representation in order to get rid of the Rayleigh line [1,2]. A peptide/ 

protein band at 110-120 cm-1 in both the Far-IR and the Raman spectrum was assigned to an out-of 

plane hydrogen bond mode. DFT calculations were performed on hydrogen bonded amide model 

systems. The mode might drive conformational changes in proteins and thus be of importance for 

protein folding and/or denaturation. In the R( ν) -representation a water band at 180 cm-1 was 

assigned to the presence of water with a tetrahedrical hydrogen bond conformation [2]. This band 

is significant for the presence of water with a bulk like structure like that in liquid water. Thus the 

180 cm-1 band can be used to monitor the presence of water with a liquid water like structure, i.e. 

water non-hydrogen bonded to proteins [2]. The FT-Far-IR synchrotron spectra are very sensitive 

to the presence of low water concentrations in water/lysozyme mixtures, whereas the Raman 

spectra are useful at higher water concentrations. Thus the two techniques are complimentary in 

studies of water/protein interactions in a wide range of water concentrations. The ATR FT-Far-IR 

technique was tested in a study of full thickness pig ear skin. The low-wavenumber Raman feature 

in the R( ν) -representation was used to characterize water in skin samples from humans, pig ears, 

hairless Guinea pigs and mice [3]. Changes in water structure by freezing and thawing of skin 

biopsies can be followed [3]. Malignant and benign human skin tumours show a higher content of 

water with a bulk like structure in the malignant case [3]. The mechanism of penetration of 

dimethylsulfoxide, DMSO, through epidermal tissue from pig ear skin involved a binding of 

DMSO to free bulk water [4]. 

References 

[1] S.E.M. Colaianni, O.F. Nielsen, J. Mol. Struct. 347, 267-281 (1995). 

[2] O.F. Nielsen,C. Johansson, K.L. Jakobsen, D.H. Christensen, M.R. Wiegell, T. Pedersen, M. Gniadecka, H.C. Wulf, 

P. Westh, Proc. SPIE 4098, 160-168 (2000). 

[3] T.M. Greve,N. R. Andersen, K.B. Andersen, M. Gniadecka, H.C. Wulf, O.F. Nielsen “Biomedical Aspects of Water 

Structure in Human and Animal Skin: A Near Infrared-Fourier Transform-Raman Study”, in New Approaches in 

Biomedical Spectroscopy, edited by K. Kneipp, R. Aroca, H. Kneipp, E. Wentrup-Byrne, Washington DC: American 

Chemical Society, ACS Symposium Series 963, 30-40 (2007). 

[4] T.M. Greve, K.B. Andersen and O.F. Nielsen, Spectroscopy 22, 405-417 (2008). 

62


Histopathological characterization of skin cancers 

using infrared micro-imaging 

E. LY 1 , O. PIOT 1 , A. DURLACH 2 , N. CARDOT-LECCIA 3 , J-P. ORTONNE 4 , P. BERNARD 5, 6 AND M. 

MANFAIT 1 . 

1. Unité MéDIAN, CNRS UMR 6237 MEDyC, Université de Reims- 

Champagne Ardenne, Faculté de Pharmacie, IFR 53, France 

2. Laboratoire Pol Bouin, CHU Maison-Blanche, Reims, France 

3. Laboratoire central d'anatomie pathologie, Hôpital Pasteur, CHU de 

Nice, France 

4. Service de Dermatologie, Hôpital de l'Archet 2, CHU de Nice, France 

5. Laboratoire de Biochimie et de Dermatologie, CNRS UMR 6237 

MEDyC, Université de Reims-Champagne Ardenne, IFR 53, France 

6. Service de Dermatologie, CHU Robert Debré, Reims, France 

Skin cancer is the most common form of human cancer and its incidence is on the rise [1]. It 

includes non-melanoma skin cancers (NMSC) and malignant melanoma (MM). The diagnosis of 

skin cancer is based on the morphological evaluation of the lesion, which requires the expertise of 

a dermatopathologist. Fourier Transform Infrared (FTIR) imaging is emerging as a promising 

objective and innovative tool for the characterization of tissue samples. Infrared (IR) spectra probe 

intrinsic molecular composition and interactions which are characteristic of the histopathological 

state of the tissue, and can be considered as real tissue-specific spectroscopic fingerprints. Contrary 

to conventional hematoxylin-and-eosin (HE) staining, spectral imaging can be performed directly 

on archival fixed and paraffin-embedded tissues. For this purpose, we have developed a 

multivariate approach (Principal Component Analysis, Clustering) for the direct analysis of 

paraffin-embedded samples. Highly contrasted color-coded images were generated and compared 

to tissue architecture from HE stained sections [2]. First, this methodology was applied on a large 

set of NMSC samples in order to create a spectral library. Based on this information, a predictive 

diagnostic algorithm was built, and tested on unknown samples. This approach permitted the 

precise location and characterization of tumor areas in non-melanoma skin cancers, in agreement 

with the histology as revealed by standard staining of the biopsy section [3]. Second, the IR 

analysis of different subtypes of cutaneous melanoma revealed automatically intra-tumoral 

heterogeneity which was closely associated with some dermatopathological parameters such as 

ulceration, Breslow thickness and poor prognosis. Taken together these results, FTIR imaging 

appears as a label-free, rapid and automated technology to help in the diagnosis of skin cancer. 

References 

[1] Amercian Cancer Society, Cancer Facts and Figures 2007, Atlanta: American Cancer Society (2007) 

[2] E. Ly, O. Piot, R. Wolthuis, A. Durlach, P. Bernard, M. Manfait, Analyst 133, 197-205 (2008) 

[3] E. Ly, O. Piot, A. Durlach, P. Bernard, M. Manfait, Analyst, DOI : 10.1039/B820998G (2009) 

63


Investigation of the Raman spectra of hemozoin 

T. FROSCH 1,2 AND J. POPP 1,2 

1. Institut für Physikalische Chemie, Universität Jena, Helmholtzweg 4, D-07743 Jena, Germany, 

torsten.frosch@uni-jena.de 

2. Institut für Photonische Technologien, Albert-Einstein-Straße 9, D-07745 Jena, Germany 

Malaria is one of the most devastating infectious diseases on earth and resistances against key 

drugs arise on a global scale. However, the molecular mode of action of those drugs is not well 

understood. Raman spectroscopy has unique potential for an identification of biological hematintargets 

and an elucidation of drug-target-interactions in living cells. 

Raman micro-spectroscopy was applied for an in situ localization of the malaria pigment hemozoin 

in Plasmodium falciparum infected erythrocytes. These in situ Raman signals of hemozoin were 

compared to Raman spectra of extracted hemozoin, the synthetic analogue ß-hematin as well as to 

hematin and hemin. Raman spectra of the hemozoin dimer were calculated ab initio (DFT) for the 

first time and used for an assignment of the experimentally derived Raman bands. This knowledge 

of the underlying normal modes is very useful for a thorough interpretation of the Raman bands 

which are influenced by ππ−stacking to the antimalarial drugs. Morphology sensitive low 

wavenumber modes of hemozoin were selectively enhanced with help of excitation wavelengths at 

633 nm and 647 nm. The mode at 343 cm -1 in the Raman spectrum of hemozoin, is strongly 

enhanced with λexc. = 647 nm and is represented by a combined, symmetric doming mode of the 

two hematin units in the hemozoin dimer. The enhancement of this vibration is stronger in the 

resonance Raman spectrum of hemozoin compared with less crystalline ß-hematin. The low 

wavenumber modes are also strongly enhanced in the resonance Raman spectra of a very 

crystalline sample of hemin and are less pronounced in the spectra of an amorphous sample of 

hematin. The selective relative resonance enhancement of the morphology sensitive Raman modes 

of hemozoin is reflected by absorption bands in the UV-VIS-NIR spectrum. Resonance Raman 

micro spectroscopy with λexc. = 647 nm was shown to have great capabilities to probe the 

morphology of hematin samples. 

References 

Fig. 1 – Dimeric unit cell of the Malaria pigment hemozoin. 

[1] Frosch, T.; Koncarevic, S.; Zedler, L.; Schmitt, M.; Schenzel, K.; Becker, K.; Popp, J., J. Phys. Chem. B (2007), 

111, 11047; 

[2] Frosch, T.; Meyer, T.; Schmitt, M.; Popp, J., Anal. Chem. (2007), 79(16) 6159-6166 

[3] Puskar, L.; Tuckermann, R.; Frosch, T.; Popp, J.; Ly, V.; McNaughton, D.; and Wood, B. R., Lab on a Chip (2007), 

7, 1125-1131 

[4] Frosch, T.; Popp, J., J. Mol. Struct. (2009), 924-926, 301; 

[5] Frosch, T.; Koncarevic, S.; Becker, K.; Popp, J., Analyst (2009) DOI:10.1039/B821705J; 

64


Automated atmospheric pressure ionization mass 

spectrometry imaging platform 

M. VOLNY 1 , M. STROHALM 1 , V. VIDOVA 1,2 , G. KRUPPA 1 , K. LEMR 1,2 , J. POL 1,3 AND V. 

HAVLICEK 1,2 

1. Institute of Microbiology, Videnska 1083, 142 20 Prague 4, Czech Republic 

2. Palacky University, Faculty of Science Tr. Svobody 8, 771 46 Olomouc, Czech Republic 

3. University of Helsinki, P.O. Box 56, FI-00014, University of Helsinki, Finland 

We present labmade automated atmospheric pressure (AP) mass spectrometry (MS) imaging 

platform coupled to FTICR spectrometer. The interest in atmospheric pressure desorption 

ionization techniques in MS was triggered by the introduction of Desorption electrospray 

ionization (DESI), which allows for the analysis of surfaces in the ambient environment. Similar 

techniques based on modified setup and principles have emerged since DESI introduction. One of 

them, Desorption atmospheric pressure photoionization (DAPPI), which is based on efficient 

thermal shock desorption of nonpolar compounds and subsequent photoionization, was used in 

this work as complimentary to DESI. APMS imaging is the logical extension of AP techniques; this 

is the first example of DAPPI imaging and one of only few reports of high resolution DESI 

imaging. By DESI we analyzed mouse brain and kidney sections and obtained 2D chemical 

distribution of different phospholipid species with high mass accuracy and resolution. The results 

were compared with MALDI and Raman Spectroscopy imaging of tissues. DAPPI is suitable for 

less polar analytes than DESI and it was successfully used for instance to obtain chemical images 

of nonpolar compounds in plant leaves. Some analytes (e.g. cholesterol in tissues) were detected 

by both ionization techniques but overall the techniques acted as complementary. In our 

experiments, DAPPI did not provide comprehensive information about lipids in tissues, 

something that DESI is good at, but on the other hand it could for instance detect aflatoxins from 

plant attacked by a fungus, which DESI failed to ionize. We also utilized both techniques to image 

wing surfaces of different insect species. 

65


Solubilization and Determination of Ketoconazole in micellar 

solution of polyethylene glycol 400. 

NEETI NEMA AND ARCHNA PANDEY 

Department of Chemistry, Dr. Hari Singh Gour University, 

Sagar (M.P.) 470 003 INDIA 

e-mail : archnapandey@indiatimes.com 

The purpose of present study was to investigate the effect of Polyethylene glycol (PEG) 

upon the solubility of antifungal drug Ketoconazole (KTCZ). KTCZ is water insoluble drug. For 

water insoluble drugs difficulties are usually encountered in selecting dissolution medium with a 

good discriminating power. In the present study, a dissolution medium based on solubility data is 

developed for KTCZ. The solubility of KTC2 in various fluids such as purified water (pH 6.4), 

cetyl trimethyl ammonium bromide (CTAB), sodium lauryl sulphate (SLS), Polyethylene glycol 

(PEG) and also in 0.1 M sulphuric acid containing SLS, CTAB, PEG (at their critical micellar 

concentrations (CMC) values) was determined. The solubility of KTCZ is increased by 2.51 fold in 

PEG + purified water system. The surprising increase in solubility of same drug by 7.29 fold is 

found in acidic PEG i.e. PEG + 0.1 M H2SO4 system. Polymeric surfactants (PEG 400) form 

nanoscopic core-shell structues above the critical micellar concentrations. The hydrophobic drugs 

while the hydrophobic part serve as reservoirs for hydrophobic drugs while the hydrophilic part 

serves as interface between the bulk aqueous phase and the hydrophobic domain. This unique 

architecture enables polymeric micelles to serve as nanoscopic depots or stabilizers for poorly 

water-soluble compounds. A new spectrophotometric method was also proposed for the 

determination of KTCZ in pure form and pharmaceutical formulation. This method is based on the 

redox-complexation reactions, which proceed in the ketoconazole, ammonium metavanadate and 

PEG system and form a red-brown coloured complex with absorption maxima of V[V] at 375 nm. 

A linear calibration graph was obtained between 1.06 μg/ml - 42.5 - μg/ml of KTCZ. The results of 

analysis have been validated statistically and by recovery studies. The proposed method is simple 

rapid, and sensitive. 

66



September 1 st Tuesday 

67


68


Primary events in the photo-dissociation of oxy-hemoglobin 

T. KOBAYASHI 1,2,3,4 , A. YABUSHITA 1 

1. Department of Electrophysics, National Chiao-Tung University, 

Hsichu 300, Taiwan 

2. ICORP, JST, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan 

3. Department of Applied Physics and Chemistry and Institute for Laser 

Science, University of Electro-Communications, 1-5-1, Chofugaoka, 

Chofu, Tokyo, 182-8585, Japan 

4. Institute of Laser Engineering, Osaka University, 2-6 Yamada-oka, 

Suita, Osaka 565-0971, Japan 

The growing number of data on heme proteins in the ultrafast time domain has revealed 

complicated photophysics of the heme that are sensitive to both the structure of the protein and to 

the ligand (CO, O2, and NO) (1). Champion et al. studied (2) the excited-state photophysics of 

hemoglobin (Hb), myoglobin (Mb), and protoheme and the effects of the ligands CO, O2, and NO 

on the excited-state photophysics. In this work, ultrafast time-resolved pump-probe signal was 

observed in visible spectral range using an ultrashort visible laser pulse. Broad spectral width of 

visible laser pulse enabled us to observe the pump-probe signal with a broadband range. A 

broadband multi-channel detector array was used to obtain the pump-probe signal at all of the probe 

frequencies simultaneously. In the simultaneous measurement at many probe wavelengths, we 

could obtain ultrafast spectral change after the photo-excitation of oxy-hemoglobin only with a 

short measurement time, avoiding laser damage on the sample. The spectra of the excited species in 

the process of ultrafast photodissociation was determined for Hb * and HbII* 

in the spectral range of 

I 

13914 cm-1 (719 nm) and 19109 cm-1 (523 nm) for the first time in the visible range. Also the time 

constant of the primary process was determined for the first time. The difference spectra ΔA in the 

delay time region of 0-100fs averaged over 10fs was studied to observe ultrafast spectral change. 

Ultrafast spectral change found in the measurement result reflects ultrafast photo-dissociation 

process of oxy-hemoglobin. 

References 

ΔA 

- 0.015 

- 0.020 

- 0.025 

- 0.030 

- 0.035 

14000 14500 15000 15500 16000 16500 17000 17500 18000 

probe frequency (cm -1 ) 

69 

5fs 

15fs 

25fs 

35fs 

45fs 

55fs 

65fs 

75fs 

85fs 

95fs 

Fig. 1 – Difference absorbance spectra averaged over 10 fs at 

different delay times. 

[1] D. A. Chernoff, R. M. Hochstrasser, A. W. Steele, Proc. Natl. Acad. Sci. USA 77, 5606-5610 (1980). 

[2] J. W. Petrich, C. Poyart, J. L. Martin, Proc. Natl. Acad. Sci. USA 27, 4049-4060 (1988).


UV Resonance Raman spectroscopic study of tyrosine 

in the TTR(105-115) peptide 

G. PIERIDOU AND S. C. HAYES 

Dept. of Chemistry, University of Cyprus, P. O. Box 20537, 1678, Nicosia, 

Cyprus 

Understanding the various interactions between amino acid residues or backbone groups that 

determine protein structure, involves probing the local environment they experience upon folding. 

As the structure of a protein in solution might differ from that in the solid state, where it is usually 

studied by x-ray diffraction, it is critical to possess a tool that provides insights on the structure in 

the native environment. We will present our efforts in characterizing the conformation of the 11residue 

peptide TTR(105-115) (YTIAALLSPYS) in solution using UV Resonance Raman 

spectroscopy. TTR(105-115) is part of the sequence of the human amyloid-forming protein 

transthyretin, corresponding to a naturally-occurring β-strand in the crystal structure of the 

protein. This peptide fragment has been shown to form ordered amyloid fibrils in vitro, 

constituting it an ideal model for the study of fibril formation in general. Our present studies have 

provided insights on the conformation of this peptide in different environments that serve as a 

first step in understanding the structural changes that lead to fibril formation. We focused our 

efforts on the characterization of the environment experienced by tyrosines in the peptide, since 

tyrosine has been reported repeatedly in the past, as part of UVRR studies, to be a good probe for 

investigating the local environment of a protein. Specifically, vibrational bands associated with the 

phenolic ring seem to be particularly sensitive to the change in local environment and particularly 

to hydrogen bonding of the phenolic OH.[1,2] Comparison of the pKa determined for the tyrosine 

side-chains in the peptide to the value for aqueous Tyr, along with various spectral observations, 

suggest that the two Tyr residues in the peptide probe two distinct microenvironments, with 

Tyr105 exposed to the solvent and Tyr114 hydrogen-bonded to either the backbone or to other 

residues’ side chains. As fibrils of TTR(105-115) have been shown to form in vitro after incubation 

at temperatures around 37 o C,[3] we performed temperature-dependent studies probing the 

tyrosine environment in the peptide. These studies demonstrated an increase in the absolute 

Resonance Raman cross sections of the tyrosine side chain vibrations with an increase in 

temperature, suggesting that the phenolic ring experiences an increasingly hydrophobic 

environment as it approaches the fibrillization temperature regime. 

References 

[1] Z. Chi X. G. Chen, J. S. W. Holtz, S. Asher, Biochemistry 1998, 37, 2854. 

[2] P. G. Hildebrandt, R. A. Copeland, T. G. Spiro, J. Otlewski, M. J. Laskowski, F. G. Prendergast, Biochemistry 1988, 

27, 5426. 

[3] C. P. Jaroniec, C. E. MacPhee, N. S. Astrof, C. M. Dobson,. R. G. Griffin, Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 

16748. 

70


Rapid-scan / step-scan FTIR difference spectroscopy applied to 

photosynthetic reactions: new insights by multiple experiment 

design and tailored multivariate analysis techniques 

A. MEZZETTI 1,2 , L. BLANCHET 2,3 , C. RUCKEBUSCH 2 , A. DE JUAN 3 AND W. LEIBL 1 

1. SB2SM, URA CNRS 2096, IBITeC-S, DSV, Bat 532, CEA-Saclay, France 

2. LASIR UMR CNRS 8516, Université Lille 1, Villeneuve d’Ascq, France 

3. Departament de Química Analítica, Universitat de Barcelona, Diagonal, 647, 08028 Barcelona, Spain 

Time-res. difference FTIR is a powerful technique to study biochemical processes. We studied 

photosynthetic systems where light induces processes like electron transfer (ET), energy transfer, 

proton transfer, or cofactor displacement [1-6 & refs. therein]. The ability of time-res. FTIR to 

record simultaneously absorbance changes over a wide range of frequencies has permitted to 

follow different events and to get information on their possible coupling (e.g. between H + and e - 

transfer). An interdisciplinary approach has been applied, combining different time-res. FTIR 

techniques, site-directed mutagenesis, tailored chemometric analysis, kinetics simulations and DFT 

calculations. Whereas step-scan FTIR (t. res. 5 μs) has allowed to study the primary photophysical 

processes [2], rapid-scan FTIR (t. res. 25 ms) has been used to follow the H +-coupled ET leading to 

the formation and release of ubiquinol QH2 [1, 3-6]. This topic has been recently the object of 

controversy [7,8]. Results have also shown that QH2 formation entails a perturbation of the 

membrane structure. The development of a specific chemometric approach (hard-soft multivariate 

curve resolution applied to difference spectra [1,4]) enabled a detailed kinetic analysis and 

provided pure spectra corresponding to different biophysical events. The results are discussed in 

the framework of the current knowledge of the structure of the photosynthetic apparatus. 

References 

Fig. 1 – time res. FTIR differ. spectra of Rb spharoides membranes 

under light showing QH2 formation, protein rearrangement and 

membrane perturbation 

[1] L. Blanchet, C. Ruckebusch, A. Mezzetti, J.P. Huvenne, A. de Juan, J. Phys Chem. B 113, 6031-6040 (2009). 

[2] A. Mezzetti, R. Spezia, Spectroscopy Int. J, 22, 235-250 (2008). 

[3] A. Mezzetti, W. Leibl, Vibrat Spectrosc, 48, 126-134 (2008). 

[4] L. Blanchet, A. Mezzetti, C. Ruckebusch, J.P. Huvenne, A. de Juan, Anal. Bioanal. Chem, 387, 1863-1874 (2007). 

[5] A. Mezzetti, W. Leibl, Eur. Biophys. J., 34, 921-936 (2005). 

[6] A. Mezzetti, W. Leibl, J. Breton, E. Nabedryk, FEBS Letters 537, 161–165 (2003) 

[7] A. Remy, K. Gerwert, Nat. Struct. Biol. 10, 637 (2003). 

[8] J. Breton, Biochemistry 46, 4459-65, (2007). 

71


Does the disulfide bridge have an effect on the 

conformational properties of the peptide hormone 

somatostatin-14? 

B. HERNÁNDEZ 1 , C. CARELLI 1 , Y. M. COÏC 2 AND M. GHOMI 1 

1. Groupe de Biophysique Moléculaire, UFR SMBH, Université Paris 13, 74 rue Marcel 

Cachin, 93017 Bobigny cedex France 

2. Unité de Chimie des Biomolécules, URA 2128, Institut Pasteur, 28 rue du Docteur 

Roux, 75724 Paris cedex 15, France 

To emphasize the role played by the S-S bridge in the structural features of somatostatin-14 (SST- 

14) [1], we have applied our previously described experimental protocol to this peptide hormone 

[2]. Newly recorded CD and Raman spectra of this cyclic peptide and its open analogue obtained 

by Cys→Ser substitution, are presented. CD spectra of both peptides recorded in aqueous 

solutions in the 100-500 µM concentration range are strikingly similar. They reveal principally that 

random conformers constitute the major population in both peptides. Consequently the S-S bridge 

has no structuring effect at submilimolar concentrations. In methanol, the CD spectrum of 

somatostatin-14 keeps globally the same spectral shape as that observed in water, whereas its open 

analogue presents a major population of helical conformers. Raman spectra recorded as a function 

of peptide concentration (5-20 mM) and also in the presence of 150 mM NaCl, provide valuable 

conformational information. All Raman spectra present a mixture of random and β-hairpin 

structures for both cyclic and open peptides. More importantly, the presence or the absence of the 

disulfide bridge, do not seem to influence considerably different populations of secondary 

structures within this range of concentration. However, Raman spectra of SST-14 reveal a peptide 

concentration effect on the flexibility of the S-S linkage, and consequently on that of its cyclic part. 

In conclusion, although the disulfide linkage does not seem to markedly influence the SST-14 

conformational features in aqueous solutions, its presence seems to be necessary to assure the 

flexibility of the cyclic part of this peptide, and to maintain its closed structure in lower dielectric 

constant environments. 

References 

[1] M. Pawlikowski, G. Meleń-Mucha, Curr. Op. Pharmacol. 4, 608-613 (2004). 

[2] G.Guiffo Soh, B. Hernández, Y.M.Coïc, F.Z. Boukhalfa-Heniche, M. Ghomi, J. Phys. Chem. B 111, 12563-12572 

(2007). 

72


Model based pre-processing in biospectroscopy 

A. KOHLER 1,2,3 , N. K. AFSETH 1 , G. D.SOCKALINGUM 4 AND H. MARTENS 1,2,3,5 

1 Centre for Biospectroscopy and Data Modelling, Matforsk/Nofima 

Food, Ås, Norway 

2 CIGENE – Center for Integrative Genetics, University of Life Sciences, 

1432 Ås, Norway 

3 Department of Mathematical Sciences and Technology (IMT), 

Norwegian University of Life Sciences, Ås, Norway 

4 MéDIAN- Université de Reims Champagne-Ardenne, CNRS UMR6237- 

MEDyC, UFR de Pharmacie, IFR 53, 51096 Reims cedex, France. 

Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France 

5 Faculty of Life Sciences, University of Copenhagen, Denmark 

Pre-processing is an important first step of the data analysis in order to remove non-relevant 

information or to separate light scattering phenomena in spectra from chemical absorbance 1. A 

major advantage of model-based pre-processing techniques is that they allow considering light 

scattering phenomena separately from chemical information. This is important in all situations 

where light scattering phenomena are informative and need to be treated separately. This may 

avoid that they are interpreted as chemical information 1. Another advantage of model based preprocessing 

is that a priori knowledge about scattering phenomena can be build into the preprocessing 

model. 

Recently we have presented an approach for estimating and correcting Mie scattering occurring in 

infrared spectra of single cells as in synchrotron based microscopic 2. The Mie scattering can be 

modeled by approximate formulae estimated before being separated from the vibrational 

absorption using extended multiplicative signal correction (EMSC). Since the Mie scattering is 

depending non-linearly on the product of radius and refractive index of the medium/sphere 

causing it, a new method based on principal component analysis (PCA) was developed for 

estimating the Mie scattering by EMSC for unknown radius and refractive index of the Mie 

scatterer. The approach is of rather generic nature for handling spectral phenomena with a priori 

known mathematical approximation model. 

When a priori knowledge about unwanted interference phenomena is not available, it can be 

obtained from the dataset itself. Systematic patterns of unwanted methodological variations can be 

estimated from replicate spectra, modeled by a linear subspace model and implemented into 

EMSC. 3 

References 

[1] Kohler A., Zimonja M., Segtnan V., Martens H., “Data preprocessing: SNV, MSC and EMSC pre-processing in 

biospectroscopy”, in Comprehensive Chemometrics, edited by Walczak B, Tauler Ferré R, Brown S., Elsevier, in 

press. 

[2] Kohler A., Sulé-Suso J., Sockalingum G.D., Tobin M., Bahrami F., Yang Y., Pijanka J., Dumas P., Cotte M., Martens 

H., Applied Spectroscopy , 62, 259-266 (2008). 

[3] Kohler A., Böcker U., Warringer J., Blomberg A., Omholt S.W., Stark E., Martens H ., Applied spectroscopy 63 (3), 

296-305 (2009). 

73


Stemness of stem cells as determined by confocal 

Raman microspectroscopy 

V. V. PULLY 1 , A. LENFERINK 1 , V. SUBRAMANIAM 1 , C. A. VAN BLITTERSWIJK 2 , AND C. OTTO 1 

1. Biophysical Engineering, BMTI, MESA + institute for Nanotechnology 

University of Twente, Enschede 7522 ND, The Netherlands. 

2. Tissue Regeneration, BMTI, Dept. of Physics, University of Twente, 

Enschede 7522 ND, The Netherlands. 

Adult human bone marrow derived mesenchymal stem cells (hBMSC) are known for their 

stemness, wherein differentiation towards various pluripotent mesenchymal lineages such as 

bone, cartilage, muscle, neurons , hepatic tissue, and still other tissues are feasible [1]. We cultured 

hBMSCs under the influence of a range of differentiation media that provide physical and 

chemical cues to guide differentiation and assembly into osteogenic, adipogenic and myogenic 

lineages. hBMSCs differentiating towards these lineages were monitored over long time periods 

up to 30 days using a home-built confocal Raman micro-spectroscope [2]. The stemness of the cells 

could be determined with confocal Raman microscopy (CRM) from measurements at regular time 

intervals from early stage till late stage of differentiation. We conclude that CRM provides a 

powerful method for non-invasive and label-free analysis of in vitro development of tissues from 

hBMSCs all through the culture period. CRM overcomes particular disadvantages in existing 

methods such as NMR, FTIR, SEM or fluorescence staining procedures. Raman spectroscopic data 

acquired as early as day 3 of a culture period showed bio-markers for osteogenesis that defined 

early stage of hBMSC differentiation. These results were in agreement with the Raman results 

obtained after day 30 of the culture period, when stem cell pluripotency was well expressed. 

Acknowledgements 

The funding of The Dutch Program for Tissue Engineering (DPTE) through the Dutch Technology 

Foundation is gratefully acknowledged. 

References 

[1] M. F. Pittienger, A. M. Mackey, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mo DH. Frauenfelder, S. G. Sligar, P. G. 

Wolynes, Science 254, 1598-1603 (1991). 

[2] H. J. van Manen, A. Lenferink and C. Otto. Anal Chem. 80, 9576-9582 (2008). 

74


Tautomerism in 5-Bromouracil: relationships with 

other 5-haloderivatives and effect of the microhydration 

M. ALCOLEA PALAFOX 1 , V.K. RASTOGI 2 , A. GUERRERO-MARTÍNEZ 1 , G. TARDAJOS 1 , JÉSSICA 

TALAYA 1 , HITESH KUMAR 2 AND J K VATS 2 

1. Departamento de Química-Física I, Facultad de Ciencias Químicas, Universidad 

Complutense, Madrid-28040, Spain, alcolea@quim.ucm.es 

2. Department of Physics, CCS University, Meerut-250 004, India 

Uracil and its derivatives play a pivotal role in basic biological processes. 5-bromouracil (5-BrU) is 

one of the well-known uncommon nucleotide bases and has the ability to co-ordinate metal or to 

bind to tissues via metals, which interfaces with the growth of cancer cells [1,2]. The existence of 

the enol tautomer is the origin of its mutagenic property [3]. As in the uracil molecule (U), 5-BrU 

may exist in various tautomeric forms differing from each other by the position of the proton. 

Tautomers U1-U6 correspond to that of uracil notation, and they are the most important and 

studied ones, Table 1. The calculations were made with the GAUSSIAN 03 package [4]. The most 

stable is the dioxo form (U1). Tautomers U7-U10 are only characteristic of 5-XU, due to the 

X=halogen atom. Solvent interactions partly modify this gas-phase order of stability. These 

stabilities are also somewhat sensitive to both the basis set and the theoretical method used, 

showing variations within 1.5 kcal/mol. U3-U4 are very close in energy, ca. 0.5 kcal/mol. The 

shortening of N3-C4 (and N1-C6) in U3, U4 and U9 increase the quinonoid character of the ring. 

U9 is not planar. It is the only one that appears with the hydrogen on the halogen atom out-of ring 

plane, and with the uracil ring strongly deformed. Tautomers in 5-XU are more stable than in U. 

Water molecules stabilize the enol forms more than the keto ones, and increase the reactivity of both 

oxygen atoms. 

Acknowledgement.: MAP, JT, AG and GT are grateful to the UCM of Spain for financial supports 

through UCM-BSCH GR58/08 grant number 921628. 

Table 1. Gas-phase relative energies of the tautomers (Kcal/mol) in uracil and in 5-XU. 

Method U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 

Uracil: 

B3LYP/6-311++G(3df,pd) + ZPE 

B3LYP/DGDZVP + ZPE 

CCSD/6-31G**, ref. [5] 

Experimental, ref. [6] 

5-FU 

B3LYP/6-311++G(3df,pd) +ZPE 

5-BrU 

B3LYP/6-311++G(3df,pd) +ZPE 


MP2/6-31G** + ZPE 

5-IU 


References 

0 

0 

0 

0 

0 

0 

0 

0 

0 

11.12 

11.68 

11.19 

9.21 

9.72 

10.24 

9.61 

10.46 

11.53 

12.76 

12.88 

12.27 

11.92 

13.06 

13.08 

13.09 

75 

12.89 

14.47 

12.39 

19 ± 6 

11.59 

11.79 

13.29 

10.85 

13.57 

18.89 

20.12 

19.62 

22 ± 1 

16.41 

17.24 

18.57 

17.71 

18.74 

20.34 

21.63 

22.60 

19.72 

17.51 

18.71 

18.66 

18.82 

86.97 

90.54 

97.26 

79.03 

76.47 

74.82 

79.84 

70.05 

92.72 

95.80 

99.95 

[1] J.P. Henderson, J. Byun, J. Takeshita, and J.W. Heinecke, J. Biol. Chem., 278, 23522 (2003). 

[2] V.K. Rastogi, M.A. Palafox, L. Mittal, N. Peica, W. Kiefer, K. Lang, S.P. Ojha, J. Raman Spectros., 38, 1227 (2007). 

[3] J.M. Berg, J.L. Tymoczko, L. Stryer, and N.D. Clarke, Biochemistry, W.H. Freeman, New York, 2002. 

[4] Gaussian 03, Revision B.04, Gaussian Inc, Pittsburg, PA, 2003. 

[5] S. Millefiori, and A. Alparone, Chem. Phys., 303, 27 (2004). 

[6] P. Beak, and J.M. White, J. Am. Chem. Soc., 104, 7073 (1982). 

90.04 

82.52 

85.76 

90.08 

75.82


Activity of upper electron-excited states in coelenterate 

bioluminescence 

N. BELOGUROVA 1 , R. ALIEVA 2 AND N. KUDRYASHEVA 1,2 

1. Institute of Biophysics, Siberian Branch of the Russian Academy of 

Sciences, Krasnoyarsk, 660036, Russia, nbelogurova@mail.ru 

2. Siberian Federal University, Svobodny, Krasnoyarsk, 660041, Russia 

Bioluminescence is a result of chemiluminescent oxidative enzymatic reactions. The theories of 

chemiluminescence and structure of the molecules predict that the process of bioluminescence 

may involve upper electron-excited states of the bioluminescent emitter molecule. It is believed 

that organic peroxides decompose to form nπ*-states of organic substances located at carbonyl 

groups. The bioluminescent emitter is supposed to be a heterocyclic compound with high 

fluorescence yield. It is specified by the upper singlet and triplet excited states of nπ*-type (Fig. 

1) with excitation located on carbonyl groups. Generation of an excited carbonyl group of the 

similar compounds is to be followed by population of lower-energy singlet states, e.g. through 

intramolecular non-radiative transitions Tnπ*~~→Sππ*. This process is permitted as transition 

between the levels of different both orbital nature and multiplicity (El Sayed rule). Emission of 

light is the final stage of the bioluminescent process (Fig. 1). The hypothesis of activity of upper 

electron-excited states of the bioluminescent emitter was first proposed by N.S. Kudryasheva 

and D.N. Shigorin. It was experimentally confirmed for bioluminescent emitter of bacteria. 

Application of the hypothesis to bioluminescent emitters of other organisms (fireflies, 

coelenterates, etc.) is of great interest now. In this work we experimentally verified that upper 

electron-excited states are involved in bioluminescence of coelenterates as the primary excited 

states. A series of fluorescent molecules was used as foreign energy acceptors in this 

bioluminescent reaction. The fluorescent aromatic compounds – pyrene, 2-methoxy-naphtalene, 

naphthalene, and 1,4-diphenylbutadiene – were selected, with fluorescent state energies 

ranging from 26700 to 32500 cm -1. Excitation of these molecules by Forster singlet-singlet energy 

transfer from Sππ* of bioluminescence emitter and by light absorption were excluded. The weak 

sensitized fluorescence of three compounds was found in the course of the bioluminescent 

reaction. Energy of the upper electron-excited states of the bioluminescent emitter was located 

around 31000 cm -1. 

Fig. 1 – Yablonski diagram of bioluminescent emitter and exogenous fluorescent compound. 

76


FTIR microspectroscopy (MSP) for detection and 

identification of fungal phytopathogenes 

Ahmad Salman 1, Leah Tsror 2, Shaul Mordechai 3 and Mahmoud Huleihel 4 

1Department of Physics, Sami Shamoon College of Engineering , Beer-Sheva 84100, Israel. 

2Department of Plant Pathology, The Institute of Plant Protection, Agricultural Research 

Organization, Gilat Experiment Station, M.P. Negev, 85250, Israel. 

3 Department of Physics, Ben-Gurion University, Beer-Sheva 84105, Israel 

4Department of Virology and Developmental Genetics, Faculty of Health Sciences, Ben-Gurion 

University of the Negev,Beer-Sheva, Israel. 

Soil-borne fungi are considered as serious pathogens to many plants and can cause a severe 

economic damage. Early detection and identification of these pathogens is very important and 

might be critical for their control. The available methods for identification of fungi are time 

consuming and not always very specific. 

Fourier-transform infrared (FTIR) microscopy is considered to be a comprehensive and sensitive 

method for detection of molecular changes in intact cells. The advantage of FTIR 

microspectroscopy over conventional FTIR spectroscopy is that it facilitates inspection of restricted 

regions of the examined samples. 

In the present study we used FTIR microscopy as a sensitive and effective assay for the detection 

and discrimination between different species and strains of fungi. Our results showed significant 

spectral differences between the various examined fungi. These results proved the possibility of 

discrimination between these fungi on the level of strains which belong to the same species. 

Keywords: FTIR microscopy, fungal detection, fungi, spectral characteristics 

77


Observing proteins as single molecules encapsulated in 

surface- tethered polymeric nanocontainers 

T. ROSENKRANZ 1 , A. KATRANIDIS 1 , D. ATTA 1 , I. GREGOR 2 , J. ENDERLEIN 2 , M. GRZELAKOWSKI 3 , 

P. RIGLER 3 , W. MEIER 3 AND J. FITTER 1 

1. Forschungszentrum Jülich, ISB-2, Molecular Biophysics, D-52425 

Jülich, Germany 

2. III Physikalisches Institut, Universität Göttingen, D-37077 Göttingen, 

Germany 

3. Institut für Physikalische Chemie, Universität Basel, 

Klingelbergstrasse 80, CH-4056 Basel, Switzerland 

Immobilizing biomolecules provides the advantage to observe them individually for extended 

time periods, unlike in case of freely diffusing molecules in solution. In order to immobilize 

individual protein molecules, we encapsulate them in polymeric vesicles made of amphiphilic 

triblock-copolymers and tether the vesicles to a cover slide surface [1]. A major goal of this study is 

to investigate polymeric vesicles with respect to their suitability for protein folding studies. The 

fact that polymeric vesicles possess an extreme stability with respect to various chemical 

conditions is supported by our observation that harsh unfolding conditions do not perturb the 

structural integrity of the vesicles. Moreover, polymerosomes prove to be permeable to GdnHCl 

and thereby ideally suited for unfolding and refolding studies with encapsulated proteins. We 

demonstrate this with encapsulated phosphoglycerate kinase, which was fluorescently labeled 

with Atto655, a dye that exhibits pronounced photoinduced electron transfer (PET) to a nearby 

tryptophan residue in the native state. Under unfolding conditions, PET is reduced and we 

monitored for individual encapsulated proteins alternating unfolding and refolding conditions. 

References 

Fig. 1 (Left) Scheme of an individual encapsulated protein labeled 

with a fluorescent dye. (Right) Repetitive unfolding/refolding 

transitions as observed for single encapsulated proteins. 

[1] T. Rosenkranz, A. Katranidis, D. Atta, I. Gregor, J. Enderlein, M. Grzelakowski, P. Rigler, W. Meier and J. Fitter, 

ChemBioChem 10,702-709 (2009) 

78


Three dimensional collagen gels as a cell culture model 

for the study of living cells by Raman spectroscopy 

F.BONNIER, A.D. MEADE, P. KNIEF, K. BHATTACHARYA, F.M. LYNG AND H.J. BYRNE 

Focas Research Institute, Dublin Institute of Technology (DIT), Kevin 

Street, Dublin 8, Ireland (fbonnier@dit.ie) 

The study of single cells with infrared and Raman spectroscopy is an emergent topic in the field of 

“biospectroscopy” [1]. The main advantage of Raman spectroscopy is the weak signal from water, 

which offers the potential to record spectra from living cells using an immersion lens. Normally, 

the confocality of the Raman microspectrometer reduces the spectral contribution of the optical 

substrates that are used during measurements on optically thin single cells. Until now, quartz has 

been preferred due to its weak signal but also for its surface properties which allow cells to attach 

and grow, in comparison to CaF2 or ZnSe [2]. Nevertheless, dissimilarities between different 

optical substrates within in vivo cell culture result in morphological and functional changes in the 

cell [3]. In order to create an experimental model closer to the real conditions encountered by the 

cell in vivo, 3-D collagen gels have been used as a substrate for spectroscopic study of living cells. 

In this paper we demonstrate the advantages and potential of such a model for Raman 

spectroscopy using 785nm as source. The Alamar Blue fluorescence assay demonstrated an 

increase in cell viability of up to 20% of A549 and HaCaT cells in the 3-D collagen matrix in 

comparison to quartz windows. An additional advantage of the use of collagen gel instead of 

quartz windows is a significant improvement in multivariate data analysis. We have observed 

through the measurement of various Z-profiles through various cells that collagen gels give the 

same background spectral contribution regardless of Z position, making background removal in 

data pretreatment more efficient. The density of the collagen gel appears too low to contribute 

significantly to the spectra recorded, with the result that it is rendered invisible spectrally. Thus 

the background contributions are reduced to that of the water based solution (medium/NaCl) 

used to keep the cells alive during the experiments. 

References 

[1] R. J. Swain and M. M. Stevens, Biochemical Society transactions 35(Pt 3), 544-549 (2007) 

[2] F. Draux, P. Jeannesson, A. Beljebbar, A. Tfayli, N. Fourre, M. Manfait, J. Sule-Suso and G. D. Sockalingum, The 

Analyst 134(3), 542-548 (2009) 

[3] A. D. Meade, F. M. Lyng, P. Knief and H. J. Byrne, Analytical and bioanalytical chemistry 387(5), 1717-1728 

(2007) 

79


Gold nanoparticles for protein detection assays. 

GIUSEPPE CHIRICO 1 , LAURA SIRONI 1 , STEFANO FREDDI 1 , LAURA D’ALFONSO 1 , MADDALENA 

COLLINI 1 , SANDRO PALLAVICINI 2 

1. Dipartimento di Fisica, Università degli studi di Milano Bicocca. 

Piazza della Scienza 3, Milano, I-20126, Italy. 

2. Dipartimento di Chimica Generale, Universita` degli Studi di Pavia, 

Pavia, I-27100, Italy. 

Gold nanoparticles (NP) few nm in size have peculiar optical properties that can be tailored for a 

variety of applications by changing their size and shape. The close vicinity of the dye to the gold 

surface can induce fluorescence quenching or enhancement depending on the dye-gold distance 

and it can be further modified by the binding of proteins to the gold surface. The specificity of the 

protein recognition is ensured by the use of antibodies specific for the selected protein. We report 

here the characterization of complexes based on 5 and 10 nm gold NPs on which Fluoresceine 

Isothiocyanate (FITC) is bound. The dye excited state lifetime is measured on single NP or small 

aggregates by fluorescence burst analysis in standard solutions. The binding of proteins to the gold 

NPs through antigen-antibody recognition induces a decrease of the dye excited state lifetime that 

can be used to measure the protein concentration. We present titrations of BSA and p53 proteins 

up to 100 pM. The brightness of this dye allows to measure protein concentration in solution with 

an apparent limit of detection of 5 pM. Competition experiments with other globular proteins in 

solutions indicate that the present constructs allow highly selective protein detection and further 

extension to experiments in cellular extracts suggests that the proposed method could proceed to 

future clinical trials. 

80


Data processing in FTIR imaging of cells and tissues: 

towards protein secondary structure imaging 

ERIK GOORMAGHTIGH, AUDREY BÉNARD, ALLISON DERENNE AND RÉGIS GASPER, 

Laboratory for the Structure and Function of Biological Membranes, 

Center for Structural Biology and Bioinformatics, Université Libre de 

Bruxelles (ULB) CP206/2, Boulevard du Triomphe 2, B1050 Brussels, 

Belgium. 

IR spectroscopic images were recorded using an Equinox Bruker FTIR spectrometer coupled to a 

Hyperion 3000 imaging system equipped with a mercury cadmium telluride (MCT)-based focal 

plane array (FPA) detector of 64×64 pixels (Bruker Optik, Ettlingen, Germany). Images of 4096 IR 

spectra at 8 cm -1 spectral resolution were acquired by coadding 256 interferograms. All the data 

processing was carried out by the program “Kinetics” running under MatLab. The “Kinetics” 

software, previously used for FTIR spectrum processing and analysis, was extended for the 

processing of FPA-acquired FTIR data. Beyond the challenge of processing thousands of spectra 

for every single image, image analysis raises new challenges related to the variations of sample 

concentration on the same image, from the absence of sample to concentrations that are too high 

and saturate the detector. Preprocessing usually starts with scaled correction for water vapor 

absorption, baseline subtraction, normalization (usually on the amide I-amide II area), CO2 

contribution removal and possibly derivation (usually second derivative, in which case baseline 

subtraction is unnecessary). All these processing steps are performed automatically. At this stage, 

when the normalization step rescales spectra that were very weak, the resulting spectra are of very 

poor signal-to-noise ratios. In turn, a number of filters where designed to mark these pixels with 

either a signal/noise below a threshold or an absolute intensity higher or lower than specified 

thresholds. The latter action allows the marking of spectra distorted because of detector saturation. 

Different types of images can be then generated, either based on the absorbance at 1 wavenumber 

or the ratio of the absorbances at 2 wavenumbers or more advanced combination of the data. 

Typically, principal component analysis (PCA) on one or a combination of several spectral regions, 

area of bands, hierarchical or k-means cluster analysis, secondary structure content evaluated as 

described in [1] or supervised classification methods such as linear discriminant analysis (LDA) or 

supervised partial least square (sPLS). We shall illustrate here more precisely how the processing 

is performed and how imaging by secondary structure can shed light on the molecular 

determinants that allow the differentiation of sub-structures in tissues. Similarly we shall illustrate 

heterogeneity in prostate cancer cells (PC-3) in culture based on secondary structure imaging. 

References 

[1] Goormaghtigh,E., J.M.Ruysschaert, and V.Raussens. Evaluation of the information content in infrared spectra for 

protein secondary structure determination, Biophys.J. 90 (2006) 2946-2957. 

81


Nanoparticle-based SERS for biodiagnostic sensing 

J. KNEIPP 1,2 , V. JOSEPH 1,2 , A. MATSCHULAT 1,2 , AND D. DRESCHER 1,2 

1. Humboldt-Universität zu Berlin, Department of Chemistry, Brook- 

Taylor-Str. 2, 12489 Berlin, Germany 

2. BAM Federal Institute for Materials Research and Testing, Richard- 

Willstätter-Str. 11, 12489 Berlin, Germany 

As demonstrated by a number of works in the recent past, the tailoring of the plasmonic and 

surface properties of metal nanostructures for the construction of efficient substrates is the key to a 

successful application of surface-enhanced Raman scattering (SERS), in particular in biodiagnostic 

applications. Nanoparticles and nanoaggregates from gold and silver have proven as versatile 

SERS-active structures for many analytical purposes. Here we will show results obtained in the 

process of construction of optical labels and sensors for bioanalytical applications. We aim at 

constructing such labels in a way that permits efficient delivery and functionalization with target 

sequences in addition to optimum SERS enhancement. For the characterization of the properties of 

the nanoparticles and their aggregates, direct (electron microscopy) and indirect methods (UV/Vis 

absorption, dynamic light scattering, DLS, and small angle x-ray scattering, SAXS) have been used. 

The delivery of nanoparticulate SERS substrates has to be adapted to the morphology and 

ultrastructure of a biological sample. Here, we will also report on in situ generation of gold and 

silver nanostructures directly in situ, prior to the Raman experiment. Several aspects, such as the 

accessibility of “hidden” substructures, as well as the change in spectroscopic perspective due to 

the extreme localization of the SERS signals were observed when we generated nanoparticles in 

situ in complex biomatrices such as pollen outer layers [1]. Different Raman processes were shown 

to be useful for obtaining the vibrational information [2, 3]. The application of one- and twophoton-excitation 

with sensors applied to eukaryotic cells will be demonstrated. Utilization of twophoton 

excited (=hyper) Raman scattering is only possible due to the SERS process. Due to 

different selection rules it enables the observation of spectral features that cannot be found in 1-P 

Raman spectra, such as IR-active or silent modes. 

References 

[1] V. Joseph, F. Schulte, U. Panne, and J. Kneipp, submitted. 

[2] J. Kneipp, H. Kneipp, K. Kneipp, Proc. Natl. Acad. Sci. USA 103, 117149-17153 (2006). 

[3] J. Kneipp, H. Kneipp, B. Wittig, K. Kneipp, Nano Letters 7, 2819-2823 (2007) 

82


Tip-enhanced and surface-enhanced Raman 

spectroscopy of biological molecules on structured 

metallic surfaces 

L. E. HENNEMANN 1 , D. ZHANG 1 , D. BENNER 2 , AND A. J. MEIXNER 1 

1. Institute of Physical and Theoretical Chemistry, University of 

Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany 

2. Department of Physics, University of Konstanz, Universitätsstrasse 10, 

78457 Konstanz, Germany 

A custom built apertureless scanning near-field optical microscope (SNOM) [1] has been used to 

investigate several kinds of biological molecules ranging from DNA bases to double stranded 

DNA. The setup is an extended parabolic mirror based confocal microscope. We detect the 

presence of (sub)monolayer molecules by collecting their unique Raman fingerprints using either 

surface-enhanced Raman spectroscopy (SERS) or tip-enhanced Raman spectroscopy (TERS). For 

SERS, both the irregular rough edges of evaporated noble metal grids and regular arrays of gold 

nano triangles served as enhancing substrates. We compared the plasmonic properties of gold 

triangles of different sizes and inter-distances to optimize the electromagnetic enhancement with 

the 632.8 nm laser excitation. Strongly enhanced Raman spectra of adenine, acridine and calf 

thymus DNA were observed. In the case of TERS, an electrochemically etched sharp gold tip 

(approx. 20 nm tip apex diameter) is approached to the surface, acting simultaneously as an AFM 

tip for topographical measurements and as a near-field antenna collecting optical information. 

Preliminary TERS measurements of single calf thymus DNA molecules immobilized on smooth 

Au (111) surfaces will be shown. 

Fig. 1 Gold nano triangles evaporated on a glass substrate. a): Confocal optical image of the gold triangle 

array. Inset: SEM picture showing partially the gold triangles and partially the template composed of 

polystyrene particles. b): SNOM measurement of the gold triangles. Left, the topography; right, the 

simultaneously recorded optical image acquired by an avalanche photodiode. The white triangles indicate 

the near-field luminescence generated by the gold nano triangles. c): SERS spectrum of (sub)monolayer 

adenine molecules adsorbed on the gold triangles. Acquisition time: 10 s, laser power: 250 µW, source: 

632.8 nm He-Ne laser. 

Refences 

[1] M. Sackrow, C. Stanciu, M. A. Lieb, A. J. Meixner, Chem. Phys. Chem. 9, 316-320 (2008) 

83


SERS microscopy: improved nanoparticle probes and 

their application in tissue diagnostics 

M. SCHÜTZ, M. GELLNER, B. KÜSTNER AND S. SCHLÜCKER 

Department of Physics, University of Osnabrück, Barbarastraße 7, 

49069 Osnabrück, Germany. 

Surface-enhanced Raman scattering (SERS) microscopy is a novel method for the selective 

detection of biomolecules in targeted research.[1,2] This technique is based on vibrational Raman 

microspectroscopy in combination with target-specific nanoparticles. The aim is to visualize and 

also quantify the distribution of multiple target molecules such as proteins in cells and tissues. 

Advantages of SERS over existing labeling approaches include the tremendous multiplexing 

capacity, quantification and high photostability.[1] Here, our improved SERS label design will be 

presented: Silica-encapsulated self-assembled monolayers (SAMs) on tunable gold/silver 

nanoshells as SERS substrates for red laser excitation.[3] This concept combines the spectroscopic 

advantages of a SAM with the high chemical and mechanical stability of a glass shell. The 

maximum surface coverage with Raman reporter molecules on the nanoparticle surface and the 

uniform molecular orientation within the SAM leads to intense and reproducible SERS signals. 

Overall, this improved SERS label design based on Au/Ag nanoshells results in ~ 180 times 

brighter SERS signals upon red laser excitation, when compared with existing approaches based 

on single gold nanospheres and submonolayer coverage with Raman reporter molecules.[3] Using 

SERS-labeled antibodies, the selective localization of prostate-specific antigen (PSA) in the 

epithelium of prostate tissue specimens by immuno-SERS microscopy is demonstrated. [2,3] 

Fig. 1 – Improved SERS label design for red laser excitation: silica- 

encapsulated SAM of Raman reporter molecules on a Au/Ag 

nanoshell 

[1] S. Schlücker, ChemPhysChem 2009, accepted (invited review on SERS microscopy). 

[2] S. Schlücker, B. Küstner, A. Punge, R. Bonfig, A. Marx, P. Ströbel, J. Raman Spectrosc. 37, 719-721 (2006). 

[3] B. Küstner, M. Gellner, M. Schütz, F. Schöppler, A. Marx, P.Ströbel, P. Adam, C. Schmuck, S. Schlücker, 

Angew. Chem. Int. Ed. 48, 1950-1953 (2009). 

84


Surface enhanced Raman scattering for tissue 

diagnostic 

S. CÎNTĂ PÎNZARU 1 , A. FĂLĂMAŞ 1 C. A. DEHELEAN 2 , AND C. LEHENE 1 

1. Babes-Bolyai University, Faculty of Physics, Kogalniceanu 1, RO- 

400084 Cluj-Napoca, Romania 

2. Victor Babes-University of Medicine and Pharmacy, Eftimie Murgu 

Square 2, RO-300041, Timisoara, Romania 

Surface enhanced Raman scattering (SERS) has been largely employed for characterizing the 

interaction between metal surfaces and complex biomolecules, like DNA or proteins, or even 

detecting the antigen–antibody complexes at a given nanostructured surface. In order to gain the 

required enhancement to detect a non-resonant Raman target species at physiological 

concentration level, a large Raman cross-section molecular label, usually a dye species can be used 

[1, 2]. Here we present the possibility to transfer this concept in the tissue investigation. The 

availability of the amino functional groups of the cresyl violet molecule for Ag adsorption and/or 

for DNA targeting is reported. The further aim of the present work was to determine whether 

SERS spectra can be acquired from cresyl violet labeled Ag nanoparticles using FT-SERS with the 

near infrared 1064 nm laser line, less aggressive for tissues investigation. The information about 

the label stability which does not suffer from photobleaching, is further used to test the specific 

Raman signatures of the reporters buried in Sprague Dowley rat tissue to probe the skin damage 

and pathology monitoring using FT-SERS. Obtaining for the first time here the FT-SERS spectrum 

of CV using Ag nanoparticles is of particular importance in the field, since a very limited number 

of molecular species exhibit NIR-SERS with Ag colloidal nanoparticles. According to the SERS 

theory, the explanation of the present results is based on the surface plasmon resonance of the 

aggregated nanoparticles in the presence of the adsorbate, which could reach NIR. It is concluded 

that cresyl violet is adsorbed on the Ag nanostructured surface through its chromophore group 

and the exocyclic NH2 groups are less involved, thus obtaining double functionalized Ag 

nanoparticles which are extremely important because they can bind via these functions to DNA 

fragments and can be used for biomedical diagnosis and spectral tagging. Besides the characteristic 

SERS lines of the reporter, the tissue spectra display the intense lipids bands located at 2974, 2852 

and 1440 cm -1 as well as characteristic DNA bands. The band shape profiles are changed on 

passing from normal to cancerous tissue, a distinct spectral shape being noticeable. The 

photodamaged skin exhibited characteristic signal, therefore, the technique shows great promise in 

rapid diagnostic and cancer tissue monitoring. 

References 

[1] T. Vo-Dinh, F. Yan, M. B. Wabuyele, J. Raman Spectrosc. 36, 640–647 (2005). 

[2] I. Pavel, E. McCarney, A. Elkhaled, A. Morrill, K. Plaxco, and M. Moskovits, J. Phys. Chem. C 112(13), 4880-4883 

(2008). 

85


A new approach for online-monitoring of drugs in 

complex matrices with Surface Enhanced Raman 

Spectroscopy 

A. MAERZ 1 , K.R. ACKERMANN 1, D. MALSCH2, T. BOCKLITZ1, T. HENKEL2 AND J. POPP 1,2 

1. Institute of Physical Chemistry, Friedrich Schiller University Jena, 

Helmholtzweg 4, 07743 Jena, Germany 

2. Intitute of Photonic Technology (IPHT), Albert-Einstein-Straße 9, 

07745 Jena, Germany 

Raman spectroscopy is characterized by high specificity, good spatial resolution, minimal sample 

preparation and the fact that water can be used as solvent. Nevertheless it is difficult to use 

Raman spectroscopy for analytical questions due to the low sensitivity. A possibility to become 

more sensitive is the use of surface enhanced Raman spectroscopy. In literature even the 

detection of single molecule with SERS is reported [1]. In combination with a lab-on-a-chip device 

surface enhanced Raman spectroscopy is excellent applicable for quantitative online-monitoring 

of different drugs like mitoxanrone and promethazine in aqueous solutions [2]. To accomplish the 

preconditions for quantitative measurements a liquid/liquid 2-phase segmented flow is applied 

to prevent depositions of nanoparticle at the channel walls [3]. To compensate the low 

reproducibility of SERS spectra due to the dependence of the SERS activity on various properties 

of the used colloid isotopic labeled molecules are implemented as internal standard [4]. This new 

lab-on-a-chip system using an isotope labeled internal standard and liquid-liquid segmentedflow-based 

flow-through Raman detection offers the possibility for reproducible quantitative 

SERS measurements. The major challenge now is to detect drugs in matrices like blood or urine. 

The necessary sample preparation is supposed to be implemented in the lab-on-the-chip system 

to create a compact analytical tool. In this contribution first investigations concerning the 

detection of drugs in matrices will be presented. 

References 

[1] S. Nie, S. R. Emory, Science 1997, 275, 1102. 

[2] K. R. Strehle, D. Cialla, P. Roesch, T. Henkel, M. Koehler, J. Popp, Anal. Chem. 2007, 79, 1542. 

[3] K. R. Ackermann, T. Henkel, J. Popp, ChemPhysChem 2007, 8, 2665. 

[4] A. März, K. R. Ackermann, D. Malsch, T. Bocklitz, T. Henkel, J. Popp, Journal of Biophotonics 2009, 2, 232. 

86


Towards the instantaneous quantitative fluoroimaging 

drugs determination in body fluids with no added reagents 

N. STRASHENIKOV 1 , A.GERSHANIK 1 , N. PAPIASHVILI 1 , R. COHEN-LURIA 1 , Y. KALISKY 2 , 

AND A.H. PAROLA 1* 

1. Dept. of Chemistry, Ben Gurion University, P.O. Box 653 Beer Sheva, Israel 84 105, Israel 

2. Chemistry Dept, Nuclear Research Center Negev, P.O. Box 9001 Beer Sheva, 84190, Israel 

The objective of our research stems from the need to develop a simple and easy-to-operate 

monitoring technique for a rapid, sensitive and quantitative detection of drugs in body fluids, with 

no reagent added and with no need for highly qualified professional laboratory technicians. This 

will be of particular use in bed-side diagnosis and for patient monitoring in doctor’s office. An 

added value of our proposed method is aimed towards hospitals that will not require special 

clinical laboratories. Mass monitoring of say, student population, government employees, military 

personnel or drugged drivers could thus be easily monitored. This novel method should result in 

elimination of the need to send body fluid samples to a central laboratory and instead will utilize 

the internet. The newly developed Fluoroimager will be widely and freely distributed for sample 

measurement elsewhere and the collected test data will be examined by our novel neuronal 

network-pattern recognition software, against a central data bank. We obtain a 3-D fluorescence 

map of the sample under study by simultaneous recording of the fluorescence intensity at the 

various predetermined spectral range of both excitation and emission wavelengths. The 3-D 

image obtained is specific and a unique "fingerprint image" of the fluorescent sample. When 

compared with various spectra from a predetermined databank (which includes concentrationcalibration 

curves), it can accurately identify both qualitatively and quantitatively the unknown 

drugs. This is correct for samples containing drug mixtures too. The method combines highsensitivity, 

quick determination, single step measurement with no need for pre-treatment or 

addition of reagents. 

Fig. 1 – Excitation-emission three-dimensional (3D) spectra of Digoxin-diazepam 

mixture in distilled water. Similar spectra were obtained in diluted human serum. 

* To whom correspondence should be addressed at aparola@bgu.ac.il 

87


Near-Infrared Fluorophores for Biomolecule Characterization and 

Imaging Applications 

G. Patonay, G. Beckford, L. Strekowski, M. Henary and S. H. Kim 

Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA 

Near-Infrared (NIR) fluorescence has been valuable in analytical and bioanalytical chemistry. NIR 

probes and labels have been used for several applications, including hydrophobicity of protein 

binding sites, DNA sequencing, immunoassays, CE separations, etc. The NIR region (700-1100 

nm) has advantages for the spectroscopist due to the inherently lower background interference 

from the biological matrix and the high molar absorptivities of NIR chromophores. During the 

studies we report here several NIR dyes were prepared to determine the importance of the 

hydrophobicity of NIR dyes in binding to biomolecules including serum albumins. We 

synthesized NIR dye homologs containing the same chromophore but substituents of varying 

hydrophobicity. Hydrophobic moieties were represented by alkyl and aryl groups and 

hydrophilic moieties were polyethylene glycols (PEG). NIR dyes of varying hydrophobicity 

exhibited varying degrees of H-aggregation in aqueous solution; PEGylated NIR dyes had very 

little aggregation and their binding constants were smaller as well, indicating that the degree of Haggregation 

could be used as an indicator to predict binding characteristics to serum albumins. 

Typical dye structures that exhibit large binding constants to biomolecules were compared in 

order to optimize applications utilizing non-covalent interactions. Recently, our research group 

introduced bis-cyanines as NIR probes. Depending on their microenvironment, bis-cyanines can 

exist as an intramolecular dimer with the two cyanines, either in a stacked form or in a linear 

conformation in which the two subunits do not interact with each other. In its intramolecular Haggregate 

form, the chromophore has a low extinction coefficient and low fluorescence quantum 

yield. Upon addition of biomolecules the H- and D-bands are decreased and the monomeric band 

is increased, with concomitant increase in fluorescence intensity. The utility of NIR dyes as tracers 

for measuring small molecule binding to biomolecules (e.g., HSA) was demonstrated via CE using 

NIR LIF detection. Certain NIR dyes accumulate in live cells allowing for imaging applications. 

Examples for cell imaging using hydrophobic dyes will be given. In addition to hydrophobic NIR 

dyes, water soluble NIR dyes are excellent candidates for both in vitro and in vivo imaging of cells 

and organs. 

88


Examination of IgG diffusion in Controlled Pore Glass Protein 

Affinity Media with Confocal Raman Spectroscopy 

N. BIAN, S. BLAZKA, K. COTONI, AND D. BELL 

Millipore Corporation, 80 Ashby Road, Bedford, Massachusetts 01730, 

USA 

Protein A affinity chromatography has been the workhorse in downstream processing of 

monoclonal antibody [1-3]. One of the common base matrices for affinity chromatography media is 

controlled pore glass (CPG). As a relatively mature material, controlled pore glass provides 

rigidity, uniform pore size, and superior flow property. The recent breakthrough is Prosep Ultra 

Plus resin that possesses the highest binding capacity of immunoglobulin antibody, especially at 

short residence time (≤ 4 min). However, while the agarose affinity resin is well examined for its 

ligand distribution and IgG diffusion kinetics [4], the same information for controlled pore glass 

does not exist. This is partly due to the opacity of controlled pore glass in water and other common 

buffers used in confocal fluorescence studies in which agarose is transparent. The irregularity of 

controlled pore glass makes it even more mysterious and challenging to visualize. In recent years, 

the development in confocal Raman spectroscopy hardware enables researchers to “see through” 

otherwise opaque materials. We examined Prosep Ultra Plus resin using confocal Raman. 

Although we were able to obtain PrA ligand distribution directly with suitable oil, the signal to 

noise is extremely low. The data acquisition time is too long to acquire a 3-D image. To solve this 

problem, we labeled the resin with a fluorescence dye at an optimized level and were able to 

obtain good 3-D image to confirm that the PrA is evenly distributed throughout the resin. 

Encouraged by this learning, we further labeled IgG with the same fluorescence dye and observed 

its diffusion into the ProSep Ultra Plus affinity media. For resin exposed to IgG for 1 min, a clear 

diffusion front of fluorescence labeled IgG is observed. This provides us with firsthand evidence 

that IgG diffusion into CPG is similar to that of spherical media in that the outer layer of the media 

sees most of IgG. IgG is diffusion limited, most probably due to steric hindrance. Further work is 

planned to compare irregular CPG and spherical media on IgG diffusion under the same binding 

conditions at different residence time. Diffusion kinetics study will be incorporated with 

mathematic modeling to further our understanding of this widely used chromatography base 

matrices. 

References 

[1] D. Follman, and RL Fahrner. Factorial screening of antibody purification processes using three chromatographic 

steps without Protein A. J Chromatogr A.1024, 79–85 (2004). 

[2] RG. Hamilton. The Human IgG Subclasses. pp 1–75. Calbiochem- Novabiochem Corporation, Texas, (1987) 

[3] H. Wlad , A. Ballagi , L. Bouakaz , Z. Gu , JC. Janson . Rapid two-step purification of a recombinant mouse Fab 

fragment expressed in Escherichia coli. Protein Expr Purif. 22(2), 325-329 (2001). 

[4] T. Bankston, M. Stone, and G.Carta. 2008. Theory and applications of refractive index-based optical microscopy to 

measure protein mass transfer in spherical adsorbent particles. J. Chromatogr. A, 1188, 242-254, (2008) 

89


Study of melanoma cell lines invasiveness using Raman 

microspectroscopy 

N. MAINRECK 1 , O. PIOT 1 , F. ANTONICELLI 2 , M. MANFAIT 1 

1. MéDIAN, UMR CNRS 6237 MEDyC, Université de Reims-Champagne 

Ardenne, IFR 53, 51 rue Cognacq-Jay, 51096 Reims cedex, France 

2. Laboratoire de Dermatologie, UMR CNRS 6237 MEDyC, Université de 

Reims-Champagne Ardenne, IFR 53, 51 rue Cognacq-Jay, 51095 Reims 

cedex, France 

Raman microspectroscopy is an innovative biophotonic technique which can be used for the 

characterization of living cells. It allows the analysis of cellular mechanism in a fundamental 

approach but also the in vivo cell characterization in a biomedical approach. In Europe and United- 

States, melanoma incidence is in constant augmentation; doubling each decade since thirty years. 

Melanoma represents nowadays 7000 new cases per year in France. The tumoral progression 

occurs generally in two steps: the first step is an epidermic horizontal growth, the second step 

corresponds to a vertical progression towards the dermis leading to metastases formation. Some 

melanomas with high invasive potential are more aggressive and consequently of bad prognosis. 

In vitro study of melanoma cell lines with different degrees of invasive potential show different 

behaviours of tumoral invasion. Our aim is to study by Raman microspectroscopy the invasion 

mechanisms of melanoma cells on models of collagen matrix miming the extracellular matrix. We 

will compare two types of melanoma cell lines: Mewo (less invasive) and SK-MEL-28 (more 

invasive). For each, spectroscopic markers related to tridimensional progression of melanoma cells 

will be determinated both for cells and collagen peritumoral. These markers could be used for 

early diagnosis and new therapeutic targets. 

90


Synchrotron based FTIR Microscopy of single stained 

lung cells: applications in cancer diagnosis. 

J. SULÉ-SUSO 1,2 , J. PIJANKA 1 , G. D. SOCKALINGUM 3 , A. KOHLER 4 , F. DRAUX 3 , P. 

DUMAS 5 , C. SANDT 5 , D. G. VAN PITTIUS 6 , G. PARKES 6 , V. UNTEREINER 3 , AND Y. YANG 1 

1. Institute for Science and Technology in Medicine, Keele University, Guy Hilton Research Centre, 

Thornburrow Drive, Stoke on Trent ST4 7BG, U. K. 

2. Staffordshire Oncology Centre, University Hospital of North Staffordshire, Stoke on Trent, U. K. 

3. MéDIAN- Université de Reims Champagne-Ardenne, CNRS UMR6237-MEDyC, UFR de Pharmacie, IFR 

53, 51096 Reims cedex, France. 

4. Centre for Biospectroscopy and Data Modeling Nofima, Ås, Norway 

5. SOLEIL Synchrotron, BP48, L’Orme des Merisiers, 91192 Gif sur Yvette Cédex, France; 

6. Pathology Department, University Hospital of North Staffordshire, Stoke on Trent, U. K. 

It is well accepted that FTIR spectroscopy and micro-spectroscopy has a tremendous potential in 

cancer diagnosis thus reducing the workload in pathology departments. This could be, amongst 

other, as an automated analytical tool that could survey unstained tissue or cytology samples for 

the presence of cancer cells. Moreover, the technique can help to better characterise cells present in 

biopsy samples that are suggestive but not diagnostic of cancer which requires patients to undergo 

further biopsies in order to confirm the diagnosis of cancer. However, these samples are already 

stained and it becomes an important issue to assess how staining could affect the FTIR spectra of 

these cells and influence the analysis. To address this issue, we have studied, using synchrotron 

based FTIR microspectroscopy, single lung epithelial and lung cancer cells pre and post staining 

with Haematoxilin-Eosin (H & E) or Papanicolau (Pap) which are widely used in pathology 

departments. Our results show that although staining caused the presence of new peaks in the 

FTIR spectra of single cells, these did not affect amides I and II (possible biomarkers in cancer 

diagnosis) both in lung epithelial cells and lung cancer cells. Furthermore, no differences were 

found when comparing cells stained at two different centres confirming the reproducibility of this 

technique. This has important implications in the standardisation of this technique for pathological 

diagnosis. We will also discuss here the applications of FTIR microspectroscopy to study different, 

already stained, pathological samples such as cytospins and smears. 

91


Excitation of cytosine-rich nucleic acids - as seen 

through time-resolved infrared spectroscopy 

ANTHONY W. PARKER 1 , PÁRAIC KEANE 2 , IAN P CLARK 1 , MICHAL WOJDYLA 2 , GERARD W. 

DOORLEY 2 , MICHAEL TOWRIE 1 , JOHN M. KELLY 1 , AND SUSAN J. QUINN 2 

1. Central Laser Facility, STFC Rutherford Appleton Laboratory, Chilton, 

Didcot, Oxfordshire, OX11 0QX, UK 

2. School of Chemistry, Trinity College, Dublin 2, Ireland 

The nucleic acid bases have short-lived 1̟̟* excited states (


Emission lifetime study of fluorescence probes based 

on G-quadruplex oligonucleotides end-labeled with 

pyrene moieties 

B. JUSKOWIAK 1 , A. DEMBSKA 1 , T. PEDZINSKI 1 AND S. TAKENAKA 2 

1. Fac. of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 

Poznan, Poland 

2. Dept. of Applied Chemistry, Kyushu Institute of Technology, 

Kitakyushu, Japan 

Certain DNA sequences that are guanine-rich can form four-stranded structures called Gquadruplexes 

under specific cation conditions. Typically, three or four G-quartets are stacked and 

held together by π– π nonbonded attractive interactions, but formed G-quadruplexes may have 

different topological structures depending on the oligonucleotide sequence and environmental 

conditions [1]. Recently, we have developed fluorescent oligonucleotide probes based on the Gquadruplex 

scaffold for detecting potassium (PSO-Potassium Sensing Oligonucleotide) [2-5]. Two 

strategies were exploited for the transduction of metal cation binding event, the fluorescence 

resonance energy transfer (FRET) [2,3] and excimer emission of the pyrene labels [4,5]. The second 

strategy is of particular interest since it enables an insight into label-label and label/nucleobases 

interaction processes. For example, the thrombin binding aptamer (TBA) with a 

d(G2T2G2TGTG2T2G2) sequence gave efficient excimer emission in the presence of potassium [4] 

but the sensor with the human telomeric sequence d(G3(T2AG3)3) showed only quenching of the 

pyrene monomer emission without noticeable excimer contribution [5]. Here, we report steady 

state and fluorescence lifetime study of two fluorescent probes abbreviated as Py-Htelom-Py and 

Py-TBA-Py, carrying pyrene moieties at both termini and sequences of Human telomere and 

Thrombin Binding Aptamer, respectively. The effect of metal cations (sodium, potassium and 

strontium) on the photophysical processes was examined in order to elucidate factors that facilitate 

the production of excimer emission. The spectra and emission kinetics data support the 

concussions that pyrene-quadruplex conjugate has multiple conformers in solution and that the 

relative orientation of pyrene and neighboring nucleobase (guanine, adenine, thymine) plays a 

crucial role in determining both the rate of electron-transfer quenching of pyrene excited state and 

the efficiency of excimer emission. 

References 

[1] T. Simonsson, Biol. Chem. 382, 621-628 (2001). 

[2] H. Ueyama, M. Takagi, S. Takenaka, J. Am. Chem. Soc. 124, 14286-14287 (2002). 

[3] B. Juskowiak, E. Galezowska, A. Zawadzka, A. Gluszynska, S. Takenaka, Spectrochim. Acta A 64, 835-843 (2006). 

[4] S. Nagatoishi, T. Nojima, B. Juskowiak, S. Takenaka, Angew. Chem. Int. Ed. 44, 5067-5070. (2005). 

[5] H. Hayashida, J. Paczesny, B. Juskowiak, S. Takenaka, Bioorg. Med. Chem. 16, 9871–9881 (2008). 

93


DNA photodamage: Study of thyminephotodimerisation 

in a locked thymine dinucleotide 

W. J. SCHREIER 1 , J. KUBON 1 , P. CLIVIO 2 , W. ZINTH 1 AND P. GILCH 1 

1. Fakultät für Physik, LMU München, Lehrstuhl für BioMolekulare 

Optik, Oettingenstr. 67, 80538 München, Germany 

2. Institut de Chimie Moléculaire de Reims, CNRS UMR 6229, Université 

de Reims Champagne Ardenne, IRF 53, UFR de Pharmacie, 51 rue 

Cognacq-Jay, 51096 Reims Cedex, France 

UV radiation and concomitant photoreactions are one of the most important external hazards to 

DNA with implications for scientific areas ranging from molecular evolution to cancer research. 

The most abundant photolesion is the cyclobutane pyrimidine dimer (CPD) formed between two 

adjacent thymine bases with a quantum yield of the order of ~ 1%. Although the small quantum 

yield hampers the study of damage formation, we have recently traced the CPD formation in an all 

thymine DNA single strand ((dT)18) [1] by means of time resolved IR spectroscopy. This 

experiment has given evidence that IR marker bands for the CPD lesion are visible within less than 

one picosecond. A more detailed investigation is possible with the locked nucleotide model 

compound TLpTL (Figure 1) where the sugar moieties are forced into a C3' endo conformation. 

This restriction increases the quantum yield for CPD formation to about 10% [2]. By means of time 

resolved IR spectroscopy on TLpTL and two other DNA model compunds we will show that: (i) 

The initially recorded absorption changes after ~ 1 ps are due to CPD photodamage. (ii) The 

quantum efficiency of CPD formation on the few picosecond time scale equals the quantum 

efficiency reported in stationary experiments [3]. CPD photodamage formation in the investigated 

DNA constructs is thus predominantly formed via the primarily photoexcited singlet ππ* state, 

whereas the triplet channel does not play an essential role. 

References 

Fig. 1 – IR difference signal obtained after 268 nm femtosecond 

excitation of T LpT L. Arrows point to the marker bands of the CPD 

form. Structure of the locked dinucleotide T LpT L and the CPD lesion. 

[1] W. J. Schreier, T. E. Schrader, F. O. Koller, P. Gilch, C. E. Crespo-Hernandez, V. N. Swaminathan, T. Carell, W. 

Zinth, B. Kohler, Science 315, 625-629 (2007). 

[2] C. Desnous, B. R. Babu, C. McIriou, J. U. O. Mayo, A. Favre, J. Wengel, P. Cliviot, J. Am. Chem. Soc. 130, 31-31 

(2008). 

[3] W. J. Schreier, J. Kubon, N. Regner, K. Haiser, T. E. Schrader, W. Zinth, P. Clivio, P. Gilch, J. Am. Chem. Soc. 

131, 5038-5039 (2009). 

94


C`-terminal domain of nonhistone protein HMGB1 as a 

modulator of HMGB1–DNA structural interactions 

E. Chikhirzhina, A. Polyanichko, E. Kostyleva, V. Vorobyev 

1 Institute of Cytology, RAS, 4 Tikhoretsky ave., Saint-Petersburg, 194064, Russian Federation 

2 Saint-Petersburg State University, 1 Uljanovskaja st., Stary Petergoff , Saint-Petersburg, 198504, Russian 

Federation 

E-mail: chikhir@gmail.com 

The HMGB1 protein (High Mobility Group protein 1) participates in the formation of functionally 

significant DNA-protein complexes. HMGB1 protein contains two DNA-binding HMGB domains, 

demonstrating preferable binding to structural distortions in DNA and negatively charged 

disordered C`-terminal region. The latter consists of continuous sequence of dicarboxylic amino 

acids residues. We have studied the structural changes in DNA-protein complexes by CD 

spectroscopy and atomic force microscopy (AFM). We have used natural HMGB1 and 

recombinant protein HMGB1-(A+B) lacked negatively charged C`-terminal region. At low ionic 

strength (15 mM NaCl) we have observed similar behavior of both proteins upon interaction with 

DNA. At higher ionic strength (150 mM NaCl) the DNA-HMGB1-(A+B) complexes show very 

high optical activity. AFM shows, that at the low concentration of HMGB1 in the complex the 

protein is distributed along DNA in a random way. Increase of HMGB1 content leads to 

cooperative interaction and a redistribution of the bound protein molecules on DNA is observed. 

Based on the data obtained we conclude that protein-protein interactions play a key role in the 

formation of highly ordered DNA-HMGB1 complexes. In the case of HMGB1-(A+B) protein the 

AFM images can be sorted out into two different groups, attributed to the pre-cooperative and 

post-cooperative stages. It was shown that C`-terminal domain modulate the interactions of DNA 

with HMGB1 protein. The complexes of the recombinant protein HMGB1-(A+B) with DNA 

demonstrate the highly-ordered supramolecular structure. We estimate that the size of HMGB1- 

(A+B) binding site on DNA is twice bigger than that of HMGB1 protein. We suggest that the C`terminal 

domain of HMGB1 also modulates the “packing” of HMGB1 molecules on the DNA. 

This work was support by Russian Foundation for Basic Research (grants №№ 07-04-01072, 09-08- 

01119). 

95


Microwave hydration measurements of the unfolding in aqueous 

systems of proteins and synthetic proteinlike polymers 

М. M. VOROB’EV 

Institute of Organoelement Compounds, Russian Academy of Sciences, 

28 ul. Vavilova, 119991, Moscow, Russia 

Microwave irradiation at 30-40 GHz (millimeter-wave range) of aqueous systems of globular 

proteins, modified chitosans or synthetic polymers was combined with hydration measurements 

by resonance method. Hydration numbers were estimated from the difference in absorption 

between undisturbed water and water with solute, as it was attributed to the solute-induced 

reduction in water mobility. Hydration data for globular proteins were compared with data 

obtained for low-weight molecules and groups. It was found that rotational mobility of water 

molecules in the hydration shells of various kinds of solutes (groups) decreased in the following 

order: water structure breaking compounds > polar groups > unfolded proteins > globular 

proteins > non-polar groups. Bound water was shown to be a measure of macromolecular 

unfolding allowing to characterize hydrophobically driven processes like temperature-induced 

coil-to-globule transition of poly(N-isopropylacrylamide) (PNIPA) and gelation of casein micelles 

at acidification [1, 2]. The amount of bound water per number of carbons in the nonpolar groups 

increased in the following order: hydrophobically modified chitosan < globular protein (BSA) < 

hydrophobically modified polyacrylamide. The hydrophobic modification of acrylamide and 

chitosan polymers gave smaller variation in hydration than did both the coil → globule transition 

of PNIPA and the masking of nonpolar groups within protein globule. Unfolding of globular 

proteins during limited hydrolysis (proteolysis) as measured by microwave method is a 

demasking process, which is important for the regulation of proteolysis kinetics [3]. 

References 

[1] M.M. Vorob’ev, Food Hydrocolloids 21, 309-312 (2007). 

[2] M. Vorob’ev, N. Churochkina, A. Khokhlov, E. Stepnova, Macromol. Bioscience 7, 475-481 (2007). 

[3] M. M. Vorob’ev, J. Mol. Catal.: B. 58, 146-152 (2009). 

96



September 2 nd Wednesday 

97


98


Computing vibrational spectra of biomolecules by 

mixed Quantum Mechanics / Molecular Mechanics 

L. GUIDONI 1 

1. Dept. of Chemistry, Chemical Engineering and Material, Università 

degli studi de L’Aquila, Monteluco di Roio - 67040 L'Aquila 

Computational spectroscopy offers the key to link structural data to the optical, magnetic, and 

dynamical observed properties of biomolecules to unravel and rationalize the molecular 

mechanisms underlying structure/function relationships. In this respect, mixed Quantum 

Mechanics / Molecular Mechanics (QM/MM) methods have been developed to allow the study a 

portion of the system at full quantum level, whereas all the rest is treated at the classical forcefield 

level [1]. This techniques has been successfully applied to study enzyme catalytic reactions 

in situ treating the active site of the enzyme with first principles Car-Parrinello molecular 

dynamics and all the rest of the protein and solvent at the classical level [2]. Using QM/MM is 

possible to calculate and to interpret a variety of spectroscopic data such as absorption and 

fluorescence spectra, EPR, NMR, infrared spectroscopy and Raman scattering. The twofold 

advantage of QM/MM is that both temperature effects and solvent (or protein) environment can 

be explicitly considered. In the present report we will illustrate the methodology and the recent 

applications to calculate Infrared and Raman scattering intensities by first principles QM/MM 

dynamics at finite temperature. The vibrational density of states calculated along a trajectory at 

room temperature has been decomposed following the strategy reported by reference [3]. The 

decomposition allow us to assign to the peaks observed in the infrared and Raman spectra the 

corresponding “effective normal modes”, that fully include the effects of temperature and 

solvent. 

References 

Fig. 1 – Effective normal mode at 168 cm-1 of Cisplatin in water 

solution. The mode (yellow arrows) is obtained by decomposing the 

vibrational density of states of QM/MM simulations at 300 K. 

[1.] A. Laio, J. VandeVondele, U. Rothlisberger, A hamiltonian electrostatic coupling scheme for hybrid Car-Parrinello 

molecular dynamics simulations, J.Phys.Chem B, 108, 7967 (2001). 

[2.] L. Guidoni , P. Maurer, S. Piana, U. Rothlisberger, Hybrid Car-Parrinello/Molecular Mechanics Modelling of 

Transition Metal Complexes: Structure, Dynamics and Reactivity, Q.S.-A.R. 21 173-181 (2002) 

[3.] M.P. Gaigeot, M. Martinez, R. Vuilleumier, Infrared spectroscopy in the gas and liquid phase from first principle 

molecular dynamics simulations: application to small peptides, Mol. Phys, 105, 19, 2857-2878 (2007). 

99


A statistical model for protein translocation across nanopores 

A. AMMENTI 1 , F. CECCONI 2 U. MARINI-BETTOLO-MARCONI 3 AND A. VULPIANI 4 

1. Dept. of Physics University of Perugia, Via A. Pascoli , I-06123 Perugia Italy 

2. INFM-CNR Center for Statistical Mechanics and Complexity and Istituto dei Sistemi 

Complessi (CNR), Via dei Taurini 19, I-00185 Roma Italy 

3. Dept. of Physics University of Camerino and INFM-CNR 

Via Madonna delle Carceri I-68032 Camerino (MC) Italy. 

4. Dept. of Physics University La Sapienza and INFM-CNR 

P.le Aldo Moro 2, I-00185 Rome Italy. 

Transport of biopolymers across membranes is fundamental to several cellular biological process: 

such as signal exchanging, transfer of genetic information and metabolic regulation. The progress 

in the technology of molecular manipulation has made possible to conduct controlled experiments 

on translocation of polynucleotyde [1,2] and polypeptide [3,4] chains across alpha-Hemolysin 

channels and solid-state nanopores. The complete theoretical interpretations of such experiments 

is still lacking. We propose to combine Molecular Dynamics at coarse-grained level and 

appropriate drift-diffusion Smoluchowski equations [5] as a integrated statistical physics approach 

to the interpretation of translocation phenomenology. In particular, we performed Molecular 

Dynamics simulations of the translocation process of a globular protein (Ubiquitin) across a 

cylindrical nanopore. The Ubiquitin is described by simplified native-centric model to investigate 

the influence of protein structural properties on translocation mechanism. A thermodynamical and 

kinetic characterization of the process is achieved by studying the statistics of blockage times, the 

mobility and translocation probability as a function of the pulling force F acting in the pore. We 

find that the transport dynamics occurs when the force exceeds a threshold F c depending on a 

free-energy barrier that Ubiquitin has to overcome in order to slide along the channel. Such a 

barrier results from competition of the unfolding energy and the entropy associated to the 

confinement effects of the pore. We implement appropriate umbrella sampling simulations to 

compute the free energy profile as a function of the position of Ubiquitin center of mass inside the 

channel (reaction coordinate). This free-energy is then used to develop a one dimensional 

phenomenological model in the reaction coordinate which explains and reproduces the behaviour 

of the observables during the translocation. 

References 

[1] J.J. Kasianowicz, E. Brandin, D. Branton, D.W. Deamer, Proc. Natl. Acad. Sci. USA 93, 13370-3 (1996). 

[2] Li Jaili, M. Gershow, D. Stein, E Brandin, J.A. Golovchenko Nature Mat. 2, 611-15 (2003). 

[3] G. Oukhaled, J. Mathe et al. Phys. Rev.Lett. 98 158101-8 (2007). 

[4] T.C. Sutherland, Y.-T. Long et al. Nano Lett. 4 1273-7 (2004). 

[5] A.M. Berezhkovskii, M.A. Pustovoit, S.M. Bezrukov, J. Chem Phys. 116 9952-6 (2002). 

100


Respiratory proteins: "breathing motions" revealed by 

molecular dynamics simulations" 

A. ROBERTAZZI 1 , M. A. SCORCIAPINO 1,2 , M. CASU 2 , P. RUGGERONE 1,3 AND M. CECCARELLI 1,3 

1. Sardinian Laboratory for Computational Material Science SLACS 

(INFM-CNR), Cittadella Universitaria, Monserrato (CA), I-09042, Italy 

2. Dept. of Chemical Sciences, University of Cagliari, Cittadella 

Univarsitaria S.S.554 bivio Sestu, Monserrato (CA), I-09042, Italy 

3. Dept. of Physics, University of Cagliari, Cittadella Universitaria 

S.S.554 bivio Sestu, Monserrato (CA), I-09042, Italy 

In the family of respiratory proteins, hemoglobins and myoglobins have been the first to be 

crystallized in ’50 [1]. Despite the availability of 3D structures, issues regarding the microscopic 

functioning remain open, such as, for instance, the R to T switching mechanism in hemoglobin or 

the ligand escape process in myoglobin. Due to the relatively small number of residues, myoglobin 

is the suitable candidate to investigate the more general structure-function paradigm, being 

defined as the hydrogen atom of biology. In particular, myoglobin has five main ligand docking 

sites, identified with Xe-NMR [2], possibly involved in ligands migration paths [3-5]. In this 

contribution, we present standard molecular dynamics simulations performed on horse heart metmyoglobin 

with no ligand migrating inside the protein matrix. With the aim of revealing intrinsic 

internal pathways, the use of a statistical approach was applied to cavity calculation, with special 

emphasis to the major pathway, from distal pocket to Xe1. Our study points out the remarkable 

dynamical behavior of Xe4, whose “breathing motions” may facilitate migration of ligands 

through the distal region. Additionally, our results highlight a two-ways path for a ligand to 

diffuse through the proximal region, possibly allowing an alternative route in case Xe1 is occupied. 

Finally, our approach led us to the identification of key residues, such as leucines that may work as 

switches between cavities. In addition to this, preliminary results of standard (and biased) 

molecular dynamics simulations on human Hemoglobin proteins are also proposed. 

References 

[1] J. C. Kendrew, R. E. Dickerson, B. E. Strandberg, R. G. Hart, D. R. Davies, D. C. Philips, V. C. Shore, Nature 185, 

422-427 (1960). 

[2] R. Tilton, I. Kuntz, G. Petsko, Biochemistry 23, 2849-2857 (1984). 

[3] A. Cossins, M. Berenbrink, Nature 454, 416-417 (2008). 

[4] M. Brunori, Trends Biochem. Sci. 26, 209-210 (2001). 

[5] U. Flogel, M. Merx, A. Godecke, U. Decking, J. Shrader, J. Proc. Nat. Acad. Sci. USA 98, 735-740 (2001). 

101


102


POSTER CONTRIBUTIONS 

SESSION A

Palermo, August 28 – September 2, 2009 13 th ECSBM −−−− Book of Abstracts

Magnetic resonance 13 th ECSBM −−−− Book of Abstracts 

G domain movements in the tRNA modifying MnmE/GidA 

complex studied with DEER spectroscopy 

SABINE BÖHME 1 , SIMON MEYER 2 , ALFRED WITTINGHOFER 2 , 

HEINZ-JÜRGEN STEINHOFF 1 , JOHANN P. KLARE 1 

1. University of Osnabrück, Barbarastr. 7, 40976 Osnabrück, Germany 

2. Max Planck-Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany 

Guanine nucleotide binding proteins (G proteins) are a family of proteins involved in the 

regulation of many cellular processes. These proteins alternate between an inactive guanosine 

diphosphate (GDP) and active guanosine triphosphate (GTP) bound state regulated by the 

intrinsic GTPase activity [1], therefore they serve as molecular switches. The G protein MnmE is a 

multidomain GTPase conserved between bacteria and eukarya. It is a homodimer in solution 

where the highly mobile G domains face each other in various orientations but are not in close 

contact as suggested by the GDP-AlFx structure of the isolated domains. Together with GidA, 

MnmE is involved in the modification of wobble uridine bases at the first anticodon position of 

particular transfer-RNAs [2]. Here we study the GTPase-coupled rearrangements of the G domains 

by trapping the protein in various steps of its GTPase cycle by double electron-electron resonance 

(DEER) spectroscopy in combination with site-directed spin labeling (SDSL)[4-6]. Distance 

measurements show that the G domains adopt an open conformation in the nucleotide free/GDPbound 

and a open/closed two-state equilibrium in the GTP-bound state, with maximal distance 

variations of 18 Å (Figure 1). With AlFx, only the closed conformation is observed. Dimerization of 

the active sites with GDP-AlFx requires the presence of potassium, thus reflecting the requirements 

for the GTPase reaction of MnmE (Meyer et al. PLoS 2009, under review). Previous studies 

suggested that the mode of complex formation between MnmE and GidA takes place by 

generating a symmetrical α2β2 heterotetramer in which the α-helical domain of MnmE and the last 

three C-terminal helices of GidA represent the main inter-protein contact sites [3]. We were able to 

provide direct evidence that GidA modulates G domain dimerization in MnmE. Furthermore, we 

demonstrate that the potassium dependency of G domain dimerization is completely abolished in 

the presence of GidA. 

References 

[1] K. Scheffzek and M. Ahmadian (2005), Cell. Mol. Life Sci. 62 (2005), 3014-3038 

[2] A. Scrima, I.R. Vetter, M.-E. Armengod, and A. Wittinghofer, EMBO J. 24 (2005), 23-33 

[3] S. Meyer, A. Scrima, W. Versees, and A. Wittinghofer, J.Mol.Biol. 380 (2008), 532-547 

[4] H.J. Steinhoff, Biol. Chem. 385 (2004), 913-920 

[5] G. Jeschke and Y. Polyhach, Phys. Chem. Chem. Phys. 9 (2007), 1895-1910 

[6] O. Schiemann and T.F. Prisner, Q. Rev. Biophys. 40 (2007), 1-53 

PA 1 

Fig. 1 Illustration of the 

conformational changes in 

MnmE upon juxtaposition of Gdomains 

(Adapted from Scrima 

et al. (2006), EMBO Journal 

25, 2940-2951).


Resolving the folding trajectory of riboswitch aptamer 

domains at atomic resolution 

J. BUCK 1 , B. FÜRTIG 1 , J. NOESKE 1 , E. STIRNAL 1 , J. WÖHNERT 2 AND H. SCHWALBE 1 

1 Institute of Organic Chemistry and Chemical Biology, 2 Institute for 

Molecular Biosciences, Center for Biomolecular Magnetic Resonance, 

J.W. Goethe-University, Max-von-Laue-Str. 7, 60438 Frankfurt, Germany 

Riboswitch RNAs have emerged as an important example for macromolecular structural 

transitions that lead to transcriptional or translational regulation of protein expression induced by 

metabolite binding [1]. Riboswitch elements are located in the 5’-UTR of certain mRNAs and 

consist of a highly specific metabolite receptor region (aptamer domain) coupled to a 3’downstream 

sequence (expression platform). Gene expression is thought to be modulated in 

response to conformational differences between the ligand-bound and the ligand-free 

conformation of the aptamer domain. Here, static and time-resolved NMR-spectroscopic 

techniques have been applied to monitor the hypoxanthine-induced folding of two aptamer 

domains (wt and G37A/C61U-mutant) of the guanine-sensing riboswitch of the B.subtilis xptpbuX 

operon and to characterize associated RNA conformations. Initiation of the ligand-induced 

RNA folding event in situ was realized by different techniques. The laser-triggered release of the 

ligand from a photocaged derivative or a mixing technique was applied and allowed for 

subsequent NMR-spectroscopic detection of binding events with residue-specific resolution in 

real-time. Combining selective isotope labeling of the RNA with NMR filter techniques resulted in 

significant spectral resolution and allowed kinetic analysis of the buildup rates for individual 

nucleotides. The final ligand-bound state is consistent with structural data of the RNA-ligand 

complex [2, 3]. For the wt-aptamer domain, three distinct kinetic steps associated with the ligandinduced 

folding were delineated. Following initial complex encounter the ligand-binding pocket is 

formed and results in subsequent stabilization of a remote long-range loop-loop interaction. 

Incorporation of NMR data into experimentally restrained molecular dynamics simulations 

provided insight into the RNA structural ensembles involved during the conformational transition 

[4]. In contrast, in the G37A/C61U-mutant the tertiary loop-loop interactions only form in the 

presence of magnesium. This change in tertiary structure formation resulted in dramatic 

alterations of RNA ligand-binding characteristics and the associated folding trajectory of this 

mutant RNA. 

References 

[1] Mandal, M., Boese B., Barrick, J.E., Winkler W.C, Breaker R. R., Cell 113, 577-686 (2003). 

[2] Batey, R.T., Gilbert, S.D., Montagne, R.K., Nature 432, 411-415 (2004). 

[3] Noeske, J., Richter, C., Grundl, M. A., Nasiri, H.R., Schwalbe, H., Wöhnert, J., Proc. Natl. Acad. Sci. USA 102, 

1372-1377 (2005) 

[4] Buck, J., Fürtig, B., Noeske, J., Wöhnert, J., Schwalbe, H., Proc. Natl. Acad. Sci. USA 104, 15699-15704 (2007) 

PA 2


Low temperature transversal relaxation (T2) 

optimization of nitroxides and their spatial distribution 

in phospholipid membranes and detergent micelles 

R. DASTVAN, B. E. BODE AND T. F. PRISNER 

Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic 

Resonance, Goethe-University, Max-von-Laue-Straße 7, 60438 Frankfurt, Germany. 

dastvan@prisner.de 

Although most membrane proteins are purified by the use of detergents, reconstitution of 

membrane proteins into liposomes is important to identify the mechanism of action of membrane 

proteins. Many membrane proteins express their full activity only when correctly oriented and 

inserted into a lipid bilayer. 1 In order to gain structural information from membrane proteins in 

their different functional state, it is important to be able to perform the spectroscopic technique not 

only in the detergent-solubilized form but also in the reconstituted state in lipid vesicles. Pulsed 

Electron-electron Double Resonance (PELDOR) 2,3 is a powerful tool to determine long range 

distances in spin-labeled macromolecules. Its applicability to membrane proteins reconstituted 

into vesicles is so far restricted because of the much faster transversal relaxation time (T2) of spin 

labels compared to detergent micelles. In order to optimize the sample condition for the 

spectroscopy experiments with respect to relaxation time, we studied the relaxation behavior of 

spin labeled fatty acids and phospholipid analogs in phospholipid vesicles and detergent micelles 4. 

The parameters which we studied were local and total spin label concentration, deuteriation, 

temperature, presence of oxygen, membrane composition, and cryoprotectant type. Furthermore, 

we investigated the effect of spatial distribution 5,6 of spin labeled fatty acids in vesicles and 

detergent micelles. Due to the hydrophobic nature of these spin labels they are expected to be 

inhomogeneously distributed through the sample. Distance measurements on these samples have 

gathered the spatial distribution functions and their comparison with analytic models. 

References 

[1] P. Äänismaa, E. Gatlik-Landwojtowicz, A. Seelig, Biochemistry 47, 10197-10207 (2008). 

[2] A. D. Milov, K. M. Salikhov, M. D. Shirov, Fiz. Tverd. Tela 23, 975-982 (1981). 

[3] G. Jeschke, Y. Polyhach, Phys. Chem. Chem. Phys. 9, 1895-1910 (2007). And references therein. 

[4] A. Volkov, C. Dockter, T. Bund, H. Paulsen, G. Jeschke, Biophys. J. 96, 1124-1141 (2009). 

[5] A. D. Milov, R. I. Samoilova, Y. D. Tsvetkov, F. Formaggio, C. Toniolo, J. Raap, Appl. Magn. Reson. 29, 703-716 

(2005). 

[6] A. D. Milov, D. A. Erilov, E. S. Salnikov, Y. D. Tsvetkov, F. Formaggio, C. Toniolo, J. Raap, Phys. Chem. Chem. 

Phys. 7, 1794-1799 (2005). 

PA 3


Conformations of phenylalanine in the tripeptides AFA 

and GFG probed by combining MD simulations with 

NMR, FTIR, Polarized Raman, and VCD Spectroscopy 

S. PIZZANELLI 1 , C. FORTE 1 , S. MONTI 1 , G. ZANDOMENEGHI 2 , B. H. MEIER 2 , A. HAGARMAN 3 , T. J. 

MEASEY 3 AND R. SCHWEITZER-STENNER 3 

1. Istituto per i Processi Chimico Fisici, CNR, Via G. Moruzzi 1, Pisa, 56124, Italy 

2. Laboratory of Physical Chemistry, ETH-Zurich, Wolfgang-Pauli- 

Strasse 10, CH-8093 Zurich, Switzerland 

3. Dept. of Chemistry, Drexel University, 3141 Chestnut Street, 

Philadelphia, Pennsylvania 19104, USA 

Conformational properties of small, flexible peptides are a matter of ongoing interest since they 

can be considered as models for unfolded proteins. However, the investigation of the 

conformations of small peptides is challenging as they are intrinsically disordered systems. 

Motivated by its relevance for the self-aggregation of peptides and its high β-sheet propensity in 

folded proteins, the conformational distribution of phenylalanine in the tripeptides AFA and GFG 

was investigated by combining Molecular Dynamics (MD) simulations with Nuclear Magnetic 

Resonance (NMR), Fourier Transform IR (FTIR), polarized Raman and Vibrational Circular 

Dichroism (VCD) measurements. In particular MD simulations were used to obtain the mean 

values and widths of φ,ψ distributions of phenylalanine in AFA and GFG, whereas the 

experimental NOEs and spin-spin coupling constants obtained from NOESY and 1D 1H NMR 

spectra, respectively, as well as the amide I’ band profile in the isotropic and anisotropic Raman, 

IR and VCD spectra were used to obtain the thermal populations of the different conformational 

states. This analysis yielded that β-strand (β) and polyproline II (PPII) states are predominantly 

populated in both AFA and GFG, however, whereas phenylalanine exhibits a propensity for βstrand 

conformations in GFG, in AFA a higher population of the PPII state is observed. This 

indicates that a change of the neighboring residue (G→A) alters the Gibbs energy landscape of the 

guest residue (F). 

Fig. 1 – Ramachandran (φ F, ψ F) probability distribution of AFA and 

GFG in water (angles in degrees) obtained from the MD simulations. 

PA 4


Structural analysis of ErbB2 unfolded C-terminal domain. 

MIKAËL FERACCI 1 , ALI BADACHE 2 , YVES COLETTE 2 AND FRANÇOISE GUERLESQUIN 1 

1. IMR – FR88, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 

20, France 

2. Centre de Recherche en Cancérologie de Marseille, Inserm UMR 891 – 

Institut Paoli-Calmettes, 27 Bd Lei Roure 13009 Marseille, France 

Overexpression of the Erbb2 receptor tyrosine kinase in breast cancers is associated with the most 

aggressive tumours and experimental studies revealed Erbb2 as a therapeutic target: Erbb2 is able 

to confer characteristics of cancerous cells, including uncontrolled proliferation, resistance to 

aptosis and increased motility [1]. Recent clinical results demonstrated the efficacy of Erbb2targeting 

therapy, however as only a fraction of patients responds successfully to the therapy and 

that the risks of recurrence are still high, further investigations are required for an improved 

understanding of the complex network of signalling pathways underlying Erbb2-driven cancer 

progression. ErbB2 protein is a homodimer constituted of an extracellular domain targeted by 

antibodies used for efficient breast cancer therapies, a membrane bound helical domain involved 

in the homodimeristion of the molecule, and an intracellular domain constituted by a kinase 

subdomain and an unfolded C-terminal extremity [2]. The unfolded C-terminal extremity is a 275 

amino acid peptide containing five phosphorylated tyrosines involved in signal transduction. 

Various partners of these residues have been identified [3, 4] and we are currently developing an 

NMR restrained docking structural analysis of various complexes. This C-terminal extremity has a 

very high contents of proline residues is characteristic of this peptide. The importance of proline 

residues in partner recognition is one of our focuses in this study. 

References 

[1] Leahy, D. J. Adv. Prot. Chem. 68, 1-27 (2004). 

[2] Bagossi P, Horvath G, Vereb G, Szollosi J, Tozser J. Biophys J. 88, 1354-63 (2005). 

[3] Schulze, W., Deng, L. and Mann, M. Mol. Syst. Biol. 1, (2005). 

[4] Marone R, Hess D, Dankort D, Muller WJ, Hynes NE, Badache A. Nat Cell Biol. 6, 515-22 (2004). 

PA 5


Interaction of alcohol with phospholipid membrane, an 

NMR and XRD investigation on DPPC-alcohol system. 

U. WANDERLINGH 1 , S. RIFICI 1 C. CORSARO, G. D'ANGELO, V. CONTI NIBALI, A.TRIMARCHI 

1. Dipt. Di Fisica, Università di Messina, Sal. Sperone 31, Vill. S.Agata, 

98166 Messina. Italy 

The investigations on interaction between phospholipid bilayer and short-chained alcohols, are 

relevant for the potential of lipid bilayer membranes to serve as model systems 1 for studies of 

various biological processes including permeability of the plasma membrane and molecular 

mechanisms of anesthesia. Because the hydrophobic portion of an alcohol favorably interacts with 

lipid hydrocarbon chains, the polar hydroxyl group remains free to form hydrogen bonds with 

polar lipid atoms that are located near the water/lipid interface. Experiments on phospholipid 

membranes have shown that alcohols can induce an interdigitated phase and at high concentration 

even promote the assembly of some of the lipids into non-bilayer structures within the membrane 

interior. In this contribution we present an High Resolution Magic Angle Spin NMR study on 

DPPC multilamellar vescicle with different concentration of several alcohols: ethanol, propanol, 

hexanol and octanol. Typical spectra are shown in the pictures below, which are taken at 

temperatures before and after main transition from the gel to liquid phase, in the case of DPPC and 

hexanol The presence of high alcohol concentration affects the membrane transition temperature 

by shifting it to lower values (about 10° C). From an analysis of the chemical shift of 1H, one can 

observe that the alcohol mainly affect the environment of the hydrogens located in the 

phospholipid tail, by promoting some disorder in the chains portions even before the transition. 

Transition to liquid phase has also an influence on proton located in the polar head. 

References 

Fig. 1 – HRMAS spectra from pure DPPC liposomes (left) and DPPC 

with hexanol, 2:1 alcohol/lipid (rigth). Thin line corresponds to 

ordered phase, thick line corresponds to disordered phase. 

[1] D'Angelo G., Wanderlingh U.,Nibali V. C., Crupi C., Corsaro C. and Di Marco G. 'Physical study of dynamics in fully 

hydrated phospholipid bilayers', Phil. Mag. 88, 4033-4046 (2008). 

PA 6


Biomarker profiling of gilthead sea bream by means of 

1H NMR metabonomics 

F. SAVORANI 1 , G. PICONE 2 , F. CAPOZZI 2 AND S. B. ENGELSEN 1 

1. Dept. of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, 

1958 Frederiksberg C, Denmark 

2. Dept. of Food Science, University of Bologna, Piazza Goidanich 60, 47023 Cesena (FC), Italy 

This research describes a metabolic profiling study of gilthead sea bream [1] from three different 

aquaculture systems using 1H NMR spectroscopy and metabonomics [2]. A total of 54 samples 

under two different storage regimens were analyzed. The assignment of all major NMR signals of 

the hydrophilic fraction was performed for the first time for this fish. Furthermore, a 

comprehensive multivariate data analysis proved able to distinguish the fish metabolism among 

the different rearing systems and to determine whether a fish was stored or not. The state of 

energy metabolism of inosine proved to be a robust biomarker for evaluating the storage time. A 

new multivariate classification tool, interval Extended Canonical Variable Analysis (iECVA) [3], 

achieved a robust classification of the three different aquaculture systems, revealing the involved 

metabolites (Figure 1). These characterizing biomarkers yielded different results for different 

storage regimens: glycogen (stress indicator), histidine, alanine and especially glycine for the 

stored samples and mainly betaine for the fresh ones. The findings represent a step forward in 

understanding how in vivo and postmortem processes affect the total quality of the final product. 

References 

Fig. 1 – iECVA plot with insets illustrating the spectral features of 

the most discriminative biomarkers. 

[1] M. C. Monfort, “General fisheries commission for the Mediterranean”, in Marketing of aquacultured seabass and 

seabream from the Mediterranean basin, edited by the Food and Agriculture Organization of the United Nations, 

Rome, (2007) 

[2] J. K. Nicholson, J. C. Lindon, E. Holmes, Xenobiotica 29, 1181-1189 (1999) 

[3] L. Nørgaard, R. Bro, F. Westad, S. B. Engelsen, J. Chemometr. 20, 425-435 (2006) 

PA 7


Barbituric acid chloroderivatives studied by 35Cl – NQR 

spectroscopy 

H. STEC 1 , M. JADśYN 1 , J. MILECKI 2 AND B. NOGAJ 1 

1. Dept. of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland 

2. Dept. of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznań, Poland 

Pyrimidine derivatives are often similar to native structures and enhance biological activity. They 

are often used as specific enzymes inhibitors to fight bacteria, fungi and weedes [1,2]. New 

methods of gene therapy also involve different kinds of nucleobases including pyrimidine 

chloroderivaties [3]. The nuclear quadrupole resonance (NQR) spectroscopy enables a 

determination of electron density distribution in the vinicity of a quadrupole nucleus studied 

directly from the measured resonance frequency. The electron density on a chlorine nuleus can be 

correlated with the biological activity of a given compound. The substances studied were 

barbituric acid chloroderivatives. Results of the NQR study of chlorine nucleus at second and 

fourth position of pyrimidine ring were compared with 13C NMR results for carbon nucleus at 

second and fourth position, 1H NMR for hydrogen nucleus at fifth position and electronegativity 

of others substituents. Theoretical calculations of studied compounds were compared with 

experimental data and produced high correlation coefficients. Results of NQR study proved that 

the frequency of 35Cl NQR in these substances increases (and the electron density on chlorine 

nuclei rises) with increasing substituents’electronegativity, however the impact is weaker in case of 

chlorine nucleus at the second position. The ionicity of C-Cl bond increases with electronegativity 

of substituents at second and fourth position. Also, the substituent size influences NQR frequency 

by steric interaction, what is much less significant for 1H NMR shifts. 

References 

Fig. 1 – Atom numbering of studied compound. 

[1] D. C. M. Chan, C. A. Laughton, S. F. Queener, M. F. G. Stevens, J. Med. Chem. 44, 2555-2564 (2001). 

[2] J. Rebehmed, F. Barbault, C. Teixeira, F. Maurel, J. Comput. Aided. Mol. Des. 22, 831-841 (2008). 

[3] J. K. Watts, G. F. Deleavey, M. J. Damha, Drug Discovery Today, 13, 842-845 (2008) 

PA 8


Combined electron magnetic resonance and density 

functional theory study of thermally induced free 

radical reactions in fructose and trehalose single crystals 

M.A. TARPAN 1 , H. DE COOMAN 1, 2 , H. VRIELINCK 1 , E. PAUWELS 2 , M. WAROQUIER 2 , E. 

SAGSTUEN 3 AND F. CALLENS 1 

1. Department of Solid State Sciences, Ghent University, Krijgslaan 281-S1, B-9000 Gent, Belgium 

2. Center for Molecular Modeling, Ghent University, Proeftuinstraat 86, B-9000 Gent, Belgium 

3. Department of Physics, University of Oslo, P.O. Box 1048 Blindern, N-0316 Oslo, Norway 

Both as models for studying the effects of radiation on the DNA sugar unit and for applications in 

dosimetry, radiation-induced defects in sugars have in the past few decades been intensively 

studied with electron magnetic resonance (EMR) techniques, often with considerable success. 

However, irradiation generally gives rise to a large variety of free radicals, resulting in strongly 

composite Electron Paramagnetic Resonance (EPR) spectra. This complexity makes studying them 

quite a challenge. Despite considerable efforts, little is still known about the identity of the radicals 

and even less about the radical formation and transformation processes and mechanisms. At room 

temperature (RT) the primary radiation products, which may be stabilized upon low temperature 

(LT) irradiation, transform into stable radicals via multistep reaction mechanisms. While the 

species formed at LT are expected to be formed by simple processes, the molecular structure and 

geometry of the stable radicals may differ considerably from that of the intact molecule even in the 

solid state (crystals). Studying the intermediate radicals in the reactions occurring after LT 

irradiation helps elucidating the formation and identity of the stable radicals. The structural 

identification of these radicals is in most cases the result of a combination of EPR, Electron Nuclear 

Double Resonance (ENDOR) and ENDOR Induced EPR (EIE) experiments and advanced quantum 

chemistry calculations based on Density Functional Theory (DFT). In the present study a 

summary is given of the experimental EMR results obtained so far on radiation-induced radicals at 

different temperatures in fructose and trehalose single crystals and powders. “In situ” Xirradiation 

at LT (10 K) without annealing, leads to spectra strongly different from those observed 

after RT irradiation and offers the possibility to study and characterize the primary radiation 

products [1]. Performing EMR measurements on samples irradiated and/or annealed at various 

temperatures between LT (10 K or 77 K) and RT allows us to study the intermediate products, and 

such studies therefore have the potential to devise mechanistic links between the primary radicals 

and the stable radicals. In the present work, our own measurements are compared with results 

reported in the EMR literature. An outline at future experimental (EMR) and theoretical (DFT) 

research will also be given. 

References 

[1] M.A. Tarpan, E. Sagstuen, E. Pauwels, H. Vrielinck, M. Waroquier, F. Callens, J. Phys. Chem .A 112, 3898-3905 

(2008). 

PA 9


PA 

Insight into Red Abalone prismatic layer using 

MAS-SS-NMR 

TOMASZ WROBEL 1,2 

1. Université des Sciences et Technologies de Lille 1, Cité Scientifique, 

F-59655 Villeneuve d’Ascq Cedex, France 

2. Dept. of Chemistry, Jagiellonian University, Ul. Ingardena 3, 30-060 

Krakow, Poland 

The Red Abalone (Haliotis rufescens) shell is a material with remarkable mechanistic properties 

acquired by merging inorganic minerals and biological macromolecules. “The mollusk shell is a 

true composite, consisting of calcium carbonate mineral associated with biopolymers, including 

lipid assemblies, polysaccharides and proteins. In some mollusk the shell is a two layer composite 

material (Fig. 1), with each layer composed of different polymorphic forms of calcium carbonate 

(prismatic layer = calcite, nacre layer = aragonite)” [1]. The strategies aiming at characterizing the 

organic components are usually based on different extraction techniques [2]. An attempt to get an 

insight into the structure of the prismatic layer organic coating in situ was performed by Magic 

Angle Spinning Solid State Nuclear Magnetic Resonance and Fourier Transform Raman 

Spectroscopy. 

References 

Fig. 1 – Abalone shell and its two types of layer structures [1]. 

[1] J. S. Evans, Chem. Rev., 2008, 108 (11). 

[2] I. M. Weiss, Ch. Renner, M. G. Strigl, and M. Fritz, Chem. Mater., 2002, 14 (8). 

10

Multidimensional spectroscopy 13 th ECSBM −−−− Book of Abstracts 

PA 

Recording of 2D-nutation NQR spectra by random 

sampling method 

O. GLOTOVA 1 , N. SINYAVSKY 2 , M. JADśYN 1 , M. OSTAFIN 1 AND B. NOGAJ 1 

1. Dept. of Physics, Adam Mickiewicz University of Poznań, Umultowska 85, 61-614 Poznań, Poland 

2. Baltic State Academy, Molodiozhnaya str. 6, 236029 Kaliningrad, Russia 

In a conventional 2D-nutation NQR spectroscopy of spin I=3/2 nuclei the NQR signal (free 

induction decay (FID) or spin echo) is recorded for varying radio-frequency (rf) pulse length which 

is incremented by a constant step from 0 up to some maximum value tmax. Then the entire set of 

time-domain data undergoes a double Fourier transform to yield the NQR nutation spectrum with 

characteristic nutation singularities from which the asymmetry parameter η of the electric field 

gradient (EFG) tensor on I=3/2 nuclei can be extracted. This procedure, however, frequently 

appears to be a very time demanding so that, for practical reasons, its usefulness is limited. 

Moreover, for small η the nutation singularities are often not well resolved. To overcome these 

limitations we adopted a novel approach already proposed in NMR spectroscopy [2]. In this 

method the rf pulse length instead of being incremented by equal steps is taken in random from 

the range 0 to tmax. An interesting and rather non-intuitive result of modifying the original 

procedure that way is the increase of resolution of the nutation singularities and sometimes the 

improvement of the signal to noise ratio of the nutation spectrum despite the fact that the number 

of data points to acquire can be significantly decreased as compared to the original procedure. 

Accordingly, the time of the experiment decreases. Figure 1 shows this effect on the simulated 

nutation spectra of powder samples. For this purpose the analytical formulas or the 2D-nutation 

NQR spectra of spin I=3/2 nuclei are derived in our work and the condition for resolving the 

spectral singularities for small values of η is given. The aforementioned theoretical results have 

also been corroborated by us experimentally by recording the nutation 35Cl-NQR spectra for 

powder samples of potassium chlorate, chloral hydrate, cyanuric chloride and 2,6-dichloropurine 

ribonucleoside. 

Fig. 1 One-dimensional nutation NQR spectra simulated by 

numerical integration of the theoretical formulas, 

γB 1/2π=50 kHz, η=0.23, t max=400 mks, 400 data points: a) 

– rf pulse length incremented by equal steps, b) – rf pulse 

length taken as normal distribution with mean value μ=0 

and standard deviation σ=100 μs. 

References 

[1] G. S. Harbison, A. Slokenbergs, T. M. Barbara, J. Chem. Phys. 90, 5292-5298 (1989). 

[2] K. Kaźmierczuk, A. Zawadzka, W. Koźmiński, I. Zhukov, J. Biomol NMR 36, 157-168 (2006). 

11


PA 

Direct and indirect toxicological effects of carbon 

nanotubes-assessed by Raman spectroscopy 

P. KNIEF 1 , F.M. LYNG 2 , A.D. MEADE 1 , AND H.J. BYRNE 3 

1. School of Physics/Focas Research Institute, Dublin Institute of 

Technology, Kevin Street, Dublin 8, Ireland 

2. Radiation and Environmental Science Centre, Dublin Institute of 


3. Focas Research Institute, Dublin Institute of Technology, Kevin Street, 

Dublin 8, Ireland 

Raman spectroscopy has been demonstrated to have significant potential in the biosciences, 

delivering high throughput, high precision and multiplexed assays of biochemical state and 

biological function in biological species [1]. Cellular analysis using Raman spectroscopy has 

become increasingly popular in recent years with demonstrated applications ranging from disease 

diagnosis to classification of microorganisms and as a probe of molecular changes at a cellular 

level occurring as a result of external toxic exposures [2]. Carbon nanotubes have attracted 

considerable interest not only for their outstanding physical and electronic properties, promising a 

potentially vast number of applications, but also for their potential toxicological risks as 

nanoparticles [3]. In this study Raman spectroscopy is employed for the determination of carbonnanotube-mediated 

direct and indirect toxicity in human alveolar carcinoma epithelial cells 

(A549). The exposure of this cell line represents the primary pathway of exposure in humans, that 

of inhalation. Univariate and multivariate analytical techniques demonstrate the direct and 

indirect aspects of exposure to single-walled carbon nanotubes. Preliminary results exhibit a dose 

dependent response, which correlates well to previous toxicological studies [4]. Independent 

component analysis is employed to further classify and collocate cellular response as a function of 

dose and to examine differences between spectra as a function of exposed concentration. This 

preliminary study further develops of Raman spectroscopy as a probe of cytotoxicity from 

exposure to nanoparticles. 

References 

[1] Lyng, F.M., et al., Vibrational spectroscopy for cervical cancer pathology, from biochemical analysis to diagnostic 

tool. Exp Mol Pathol, 2007. 82(2): p. 121-9. 

[2] Knief, P., et al., Raman spectroscopy – a potential platform for the rapid measurement of carbon nanotubeinduced 

cytotoxicity. Analyst, 2009. 134(6): p. 1182-1191. 

[3] Dowling, A.P., et al., Nanoscience and nanotechnologies: opportunities and uncertainties in The Royal Society. 

2004. p. 11. 

[4] Herzog, E., et al., - accepted - Oxidative stress response of human lung epithelial cells upon carbon nanoparticle 

exposure depends on dispersion medium - Toxicology and Applied Pharmacology, 2008. 

12


PA 

Two-dimensional electronic spectroscopy signatures of 

the glass transition 

K. L. M. LEWIS, J. A. MYERS, P. F. TEKAVEC, F. FULLER AND J. P. OGILVIE 

Dept. of Physics and Biophysics, University of Michigan, 450 Church Street, 

Ann Arbor, Michigan 48109, USA 

Two-dimensional electronic spectroscopy (2DES) is a sensitive probe of solvation and relaxation 

dynamics on ultrafast timescales, providing detailed information about the physical origin of 

inhomogeneous line shapes. In this contribution we explore the use of 2DES for studying the glass 

transition. As a probe of the local glass environment we examine the 2DES line shape of cresyl 

violet acetate dissolved in a glass-forming solvent. In general, 2D electronic spectra correlate 

excitation and detection frequencies as a system relaxes, providing information about the 

persistence of “memory” of initial excitation frequency. Above the glass transition, the local 

environment of the chromophore changes rapidly, resulting in rapid memory loss, while below the 

glass transition the relatively static chromophore environment means that the memory persists 

over long time scales. The persistence of memory is captured by the frequency-frequency 

correlation function, and can be related to the ellipticity of the 2DES line shape [1]. We discuss the 

relative sensitivity of 2DES over other methods such as the three pulse photon echo peak shift in 

characterizing the glass transition. 

References 

Fig. 1 – Absolute value spectra of cresyl violet dissolved in a 1:1 

(v/v) mixture of water and glycerol. Both are taken at a t 2 value of 

1.5 ps. The spectrum on the left is taken above the glass transition, 

while the one on the right is below the glass transition. The large 

ellipticity for the data taken below the glass transition reflects the 

frozen environment of the chromophore. 

[1] K. Lazonder, M. S. Pshenichnikov, and D. A. Wiersma, "Easy interpretation of optical two-dimensional correlation 

spectra," Opt. Lett. 31, 3354-3356 (2006). 

13


PA 

Observation and modeling of wavepacket dynamics 

with two-dimensional electronic spectroscopy 

J. A. MYERS, K. L. M. LEWIS, P. F. TEKAVEC, F. FULLER, AND J. P. OGILVIE 

Dept. of Physics and Biophysics, University of Michigan, 450 Church Street, 

Ann Arbor, Michigan 48109, USA 

Two-dimensional electronic spectroscopy (2DES) is an emerging method that is analogous to twodimensional 

NMR spectroscopy at optical frequencies. It permits the study of coupling between 

electronic transitions, and provides detailed information about the physical origin of broad 

electronic line shapes. Recently 2DES has been shown to be a powerful tool for studying energy 

transfer in natural light-harvesting systems. Despite the wealth of information available from 

2DES, its experimental complexity has limited its applications. Many of the experimental 

challenges of 2DES can be lessened using a simple pump-probe geometry, where the two collinear 

pump pulses are created using an acousto-optic pulse shaper [1]. We have recently implemented 

this method with the use of a continuum probe, providing information over a broad range of 

frequencies, and showing that a pump-probe experiment can be readily converted to perform 

2DES measurements with the addition of a pulse-shaper. Here, we discuss the benefits of this 

method, and use the technique to study wavepacket dynamics in a simple dye system: N, N’-bis 

(2,6-dimethylphenyl) perylene-3,4,9,10-tetracarboxylicdiimide (PERY) dissolved in DMSO. We 

find that the ellipticity and peak amplitudes are modulated by a low-frequency intramolecular 

vibrational mode. This finding implies that care must be taken to include the effects of vibrational 

dynamics in interpreting line shapes in 2DES. We compare our experimental results to simulations 

that include the effects of finite pulse duration and pulse chirp and discuss future applications. 

References 

Fig. 1 – Experimental absorptive 2D spectra for PERY dissolved in 

DMSO at different waiting times [2]. The ellipticity of the peaks is 

modulated by vibrational wavepacket dynamics. 

[1] J. A. Myers, K. L. Lewis, P. F. Tekavec, and J. P. Ogilvie, "Two-color two-dimensional Fourier transform electronic 

spectroscopy with a pulse-shaper," Opt. Express 16, 17420-17428 (2008). 

[2] P. F. Tekavec, J. A. Myers, K. L. M. Lewis, and J. P. Ogilvie, "Two-dimensional electronic spectroscopy with a 

continuum probe," Opt. Lett. 34, 1390-1392 (2009). 

14


PA 

Spectroscopic imaging of Quantum dot cellular 

interaction 

L. SALFORD 1 , F. M. LYNG 2 , A. D. MEADE 1 , F. BONNIER 1 AND H. J. BYRNE 3 

1. 1. School of Physics, Dublin Institute of Technology, Kevin Street, 


2. Radiation and Environmental Science Centre, Dublin Institute of 


3. Focas Research Institute, Dublin Institute of Technology, Kevin Street, 


Quantum dots (QDs) are a diverse class of engineered nanomaterials that have great potential for 

use as agents in imaging, diagnostic and drug-delivery because of their intense and photostable 

fluorescence.[1,2] Advances in the field of nanotoxicology, however, have recently identified 

potential risks and hazards associated with exposure to QDs.[3] The main purpose of our research 

is to investigate the capabilities of a synergistic study with different techniques: cytotoxicity 

assays, confocal microscopy and vibrational spectroscopy. With the combination of these 

techniques we hope to understand the mechanisms of the interaction of QDs with biological 

systems.Fluorescence and Confocal Microscopy studies have demonstrated that internalization of 

the QDs within HaCaT keratinocytes occurs within 1 hour of exposure, and that the QDs are 

possibly retained in the lysosomes. This is confirmed by colocalization of green LysoTracker 

probes (Invitrogen, Carlsbad, CA, USA) and the QDs (Evident Technology, Troy, N.Y., USA, with 

an emission at 625 nm). Lysotracker is a fluorescent acidotropic probe for labeling acidic organelles 

in live cells.Using an excitation wavelength of 785 nm we have also observed a the two-photon 

excitation and fluorescence in QDs (emission at 625 nm), where the fluorescent emission is 

observed in the anti-Stokes Raman signal, together with the usual Stokes Raman scatter of a single 

cell. This study demonstrates preliminarily the potential for the use of QDs as lysosome Raman 

tags. QDs localize in the lysosomes and due to their intense fluorescence can be observed in the 

cell with both confocal and Raman microscopy. Raman microspectroscopy therefore offers the 

means to both localise (image) and study the chemical interaction between a nanoparticle and its 

biological environment. 

References 

[1] Gao, X., et al., In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol, 2004. 

22(8): p. 969-76. 

[2] Michalet, Quantum dots for live cells, in vivo imaging, and diagnostic. science, 2005. 

[3] Hardman, R., A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental 

factors. Environ Health Perspect, 2006. 114(2): p. 165-72. 

[4] van Manen, H.-J. and C. Otto, Hybrid Confocal Raman Fluorescence Microscopy on Single Cells Using 

Semiconductor Quantum Dots. Nano Letters, 2007. 7(6): p. 1631-1636. 

15


PA 

Fluctuating coherence length in 2D spectra of molecular 

aggregates - stochastic approach for modelling coherent 

nonlinear optical response (simulation study) 

F. ŠANDA 1 , V. PERLÍK 1 AND S. MUKAMEL 2 

1. Institute of Physics, Faculty of Mathematics and Physics, Charles 

University, Ke Karlovu 5, Prague 2, 121 16, Czech Republic 

2. Dept. of Chemistry, University of California, Irvine, CA 92697, USA 

Exciton coherence (or delocalization) length is a powerful concept for understanding the optical 

properties of molecular aggregates. It measures, how long is the part of aggregate (“fragment”) 

over which is the quantum coherence spanned after excitation. Its actual value results from the 

competition between intermolecular dipole-dipole interaction and dephasing (localization) by bath 

induced fluctuations and disorder of local site frequencies [1]. Coherent 2D photon echo 

spectroscopy provides direct measure for dynamics of fluctuation of coherence length within 

certain parameter regimes, where the fragments can be associated with particular regions of the 

spectrum [2]. We have simulated Frenkel exciton model of molecular agreggate with fluctuating 

site energies, coupling and/or dipolemoments. We used the stochastic approach [3] to bath 

induced fluctuations which is capable to correctly describe their autocorrelation timescales. 3-rd 

orderd optical response is calculated using quasiparticle representation of optical aggregate [4]. 

The signatures of the coherence length and its dynamics in both linear and 2D spectra of simple 

aggregates will be discussed. 

1.5 

1 

0.5 

References 

0 

1 1.5 2 

ω1 

J 

ω3 

J 

2.2 

2 

1.8 

1.6 

1.4 

1.2 

1 

1 1.5 2 

ω1 

J 

16 

ω3 

J 

2.2 

2 

1.8 

1.6 

1.4 

1.2 

1 

1 1.5 2 

Fig. 1 – Linear and 2D photon echo spectra of disordered tetramer. 

Higher frequencies indicate higher coherence length (here). Crosspeaks 

at longer delay time (right panel) show kinetics of fragments. 

[1] V. M. Kenkre and P. Reineker,Exciton Dynamics in Molecular Crystals and Aggregates, Springer, Berlin (1982). 

[2] F. Sanda, V. Perlik, S. Mukamel, (in preparation). 

[3] Y. Tanimura, J. Phys. Soc. Jpn. 75, 082001 (2006). 

[4] D. Abramavicius, B. Palimieri, D. V. Voronine, F. Sanda, S. Mukamel, Chem. Rev. 109, xxx (2009) 

http://dx.doi.org/10.1021/cr800268n. 

ω1 

J 

25 

20 

15 

10 

5 

0 

−5 

−10

Neutron scattering 13 th ECSBM −−−− Book of Abstracts 

PA 

EINS study of molecular motions in systems of 

biological interest 

A. BENEDETTO 1 , S. MAGAZÙ 1 , M. A. GONZALEZ 2 , F. MIGLIARDO 1,3 AND C. MONDELLI 2 

1. Dept. of Physics, University of Messina, Sperone 31, Messina, I-98166, 

Italy 

2. Institut Laue Langevin, 6 rue Jules Horowitz, Grenoble, BP 156, F- 

38042, France 

3. Laboratoire de Dynamique et Structure des Matériaux Moléculaires, 

University of Lille 1, UMR CNRS 8024, Villeneuve d’Ascq CEDEX, F- 

59655 France 

The present work is addressed to the study of the role of the instrumental resolution in Elastic 

Incoherent Neutron Scattering (EINS). The Self Distribution Function (SDF) procedure is presented 

and connected to the measured EINS intensity profile. This procedure allows to evaluate both the 

total and the partial Mean Square Displacement (MSD), through the total and the partial SDFs. The 

SDF and the Gaussian procedures are compared by applying the two approaches to EINS data 

collected by the IN13 backscattering spectrometer (ILL, Grenoble) on systems of biological interest, 

such as disaccharides, i.e. sucrose and trehalose, and lysozyme. 

17


PA 

The role of myelin proteins in degenerative diseases – A 

neutron investigation. 

W. KNOLL 1 , F. NATALI 2 , J. PETERS 1 AND P. KURSULA 3 

1. University Joseph Fourier and Institut Laue-Langevin, Grenoble, FR- 

38000, France 

2. CNR-INFM, OGG, c/o Institut Laue-Langevin, Grenoble, FR-38000, 

France 

3. University of Oulu, Oulu, Finland 

Myelin, the multilamellar, lipid-rich membranous sheath surrounding and insulating the nerve 

axons in the central nervous system (CNS) and peripheral nervous system (PNS), allows for the 

fast saltatory transmission of nerve impulses in vertebrates. It is destroyed by autoimmune 

processes in demyelinating diseases, such as multiple sclerosis (MS) or peripheral neuropathies 

(PN). The etiology of MS is affected by dynamic changes in protein-lipid interactions and lipidbased 

membrane microdomains. Two constituent parts of the myelin which account for its stability 

are the myelin basic protein (MBP) and the P2 protein. MBP is the major protein of the myelin 

membrane in the CNS and PNS, a lipid-free and intrinsically unstructured protein partitioning in 

the raft/non-raft microdomains of CNS myelin [1-2]. P2 is a basic fatty acid binding protein of the 

PNS located on the cytoplasmic side of compact myelin membranes. To investigate the influence of 

these myelin proteins on myelin stability and dynamic changes in protein-lipid interactions, we 

carried out incoherent elastic neutron scattering experiments on a simple model system consisting 

of layers of the lipids dimyristoyl L-α-phosphatidic acid (DMPA) and the myelin proteins MBP 

and P2 in a wide temperature range. 

References 

[1] R. Smith, J Neurochem 59, 1589-1608 (1992). 

[2] G. Harauz, et al., Micron., 35(7), 503-542 (2004). 

18


PA 

Quasi-elastic and elastic scattering studies of aligned 

DMPC multilayers at different hydrations 

M. TRAPP 1 , T. GUTBERLET 2 , F. JURANYI 3 , M. TEHEI 4 , T. UNRUH 5 AND J. PETERS 1,6,7 

1. Institut de Biologie Structurale, F-38042 Grenoble Cédex 9, France 

2. JCNS at FRMII, D-85747 Garching, Germany 

3. LNS, ETHZ & Paul Scherrer Institut, CH-5232 Villigen, Switzerland 

4. AINSE, University of Wollongong, NSW 2522, Australia 

5. FRM II, TUM, D-85747 Garching, Germany 

6. Université Joseph Fourier, F-38042 Grenoble Cédex 9, France 

7. Institut Laue Langevin, F-38042 Grenoble Cédex 9, France 

Lipid model membranes such as 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) serve as 

role models for their more complex counterparts in biological systems. Quasi-elastic neutron 

scattering (QENS) [1,2], inelastic neutron scattering (INS) [3] and neutron spin echo spectroscopy 

(NSE) [4] have been employed to study local as well as collective dynamics of these membranes. 

Most of these studies lack a systematic investigation of the behavior of the model membranes in 

dependence on their hydration. To probe both dynamics in the plane of the membrane and 

perpendicular to it, the samples were prepared on cleaned silicon wafers. The hydration for the 

two samples (chain deuterated DMPC-d54) was adjusted by hydrating them for pure D2O and 

from a saturated salt solution respectively, resulting in two different states of hydration (repeating 

distance d=62.5 Å for 100 % r.h. and d = 54.9 Å for 99.7 % r.h., respectively). The alignment and 

mosaicity were checked prior to the measurements for all samples by neutron diffraction. QENS 

experiments were performed at the time-of-flight spectrometer TOFTOF at FRMII in Munich 

(energy resolution: 56 µeV) in the temperature range from 5°C to 30°C to cover the main phase 

transition of DMPC which occurs around 23°C. Elastic incoherent neutron scattering (EINS) 

measurements were performed at the high momentum transfer backscattering spectrometer IN13 

(energy resolution: 8 µeV) at ILL, Grenoble. For the QENS experiment elastic incoherent structure 

factors (EISF) were extracted, which reveal that hydration has a clear influence on the mobility of 

this system and in the framework of earlier QENS [1] and backscattering [5] investigations, they 

extend our knowledge of model membrane systems. Apart from the results of these two 

measurements (TOFTOF and IN13) an outlook for further measurements (IN16) will be presented. 

References 

[1] S. König et al., J.Phys.II France, 2 (1992) 1589-1615 

[2] M.C. Rheinstädter et al., Phys. Rev. E 75 (2007) 011907 

[3] M.C. Rheinstädter et al., Phys. Rev. Lett. 93 (2004) 108107 

[4] M.C. Rheinstädter et al., Phys. Rev. Lett. 97 (2006) 048103 

[5] M.C. Rheinstädter et al., Phys. Rev. E 71 (2005) 061908 (2002) 

19

Optical spectroscopy 13 th ECSBM −−−− Book of Abstracts 

Vibrational spectroscopy investigation of 

5-methylcytosine using ab initio Hartree-Fock and 

density functional theory 

D. K. SHARMA 1 , T. RASHEED 2 , S. AHMAD 2 AND V. K. RASTOGI 1 

1. Dept. of Physics, C. C. S. University, Meerut-250004, India 

2. Dept. of Physics, Aligarh Muslim University, Aligarh-202002, India 

A clear understanding of the vibrational spectra of 5-methylcytosine (5MC) will aid considerably 

further studies of certain biological processes in a more complex biomolecular system. In the 

present investigation, quantum chemical calculations of 5MC (Fig. 1) have been performed using 

the computation package Gaussian 03W. The equilibrium geometry, harmonic vibrational 

frequencies, infrared intensities and Raman scattering activities were calculated by HF and density 

functional B3LYP [1-2] methods utilizing the 6-311G(d,p) basis set. Calculated bond lengths and 

bond angles have been compared with the experimentally determined values for cytosine [3] and 

these values were found to be in good agreement. A detailed interpretation of 5MC vibrational 

spectra is reported. The molecular visualization program GaussView 03W was used to observe 

normal modes of vibration. The results are discussed in context of assigning the normal modes of 

5MC. The scaled theoretical frequencies are in general close to the experimental ones which allow 

an unambiguous assignment of vibrational modes. Experimental frequencies for the asymmetric 

and symmetric stretching vibrations of the NH2 group are assigned at 3555 and 3435 cm -1 

respectively. Calculated and experimental frequencies have large difference which may be 

attributed to the intermolecular hydrogen bonding in the solid sample. The band observed at 1745 

cm -1 in the Raman spectrum corresponds to the stretching vibration, ν(C=O), which is found to be 

very close to the scaled DFT frequency of 1759 cm -1. The lower frequencies generally show mixed 

composition of many modes. 

References 

Fig. 1 – Molecular structure and numbering scheme for 5methylcytosine. 

[1] A.D. Becke, J. Chem. Phys. 98, 1372-1378 (1993); J. Chem. Phys. 98, 5648-5756 (1993). 

[2] C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 37, 785-789 (1988). 

[3] D.L. Barker, R.E. Marsh, Acta Cryst. 17, 1581-1587 (1964). 

PA 20


Conformational Heterogeneity of Cytochrome c Probed 

by Resonance Raman Spectroscopy as a function of pH 

M. ALESSI 1 , A. HAGARMAN 1 AND R. SCHWEITZER-STENNER 1 

Dept. of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, 19104, USA 

The oxidized state of cytochrome c is a subject of continuous interest, due to the multitude of 

conformations the protein adopts. These conformations are described as deviations from the native 

state, adopted at neutral pH and room temperature. These so-called non-native states are thought 

to be adopted during biological functions such as electron transfer, peroxidase activity and 

apoptosis. Despite numerous studies, native and non-native states of ferricytochrome c have not 

been comprehensively analyzed regarding the influence of solvent conditions on structure, 

function, and thermodynamic equilibrium. The heme group of cytochrome c can be best related to 

having D4h symmetry, with a square planar geometry. Complexes that are classified in terms of 

D4h have four types of Raman active modes, which exhibit A1g, A2g, B1g, and B2g symmetry. These 

normal modes correspond to different macrocycle deformations. In ideal D4h, the Raman modes 

would have characteristic depolarization ratio (DPR) values which are as follows: A1g (0.125), A2g 

(∞), B1g (0.75) and B2g (0.75). Deviations of DPRs from expectation values allows for insight into 

admixtures of macrocycle deformations. These deviations can be inferred from figure 1. In the 

current study, we have analyzed the high frequency (1200-1800cm -1) Soret and Q-band resonance 

Raman spectra of oxidized and reduced horse heart cytochrome c (hhc) in terms of depolarization 

ratios as a function of increasing pH. The obtained DPR values of the analyzed Raman lines 

indicate that the alkaline transitions from the native state III to non-native alkaline states IV induce 

an additional triclinic B2g deformation. The results show that as cytochrome c undergoes its 

transition from the native to non-native alkaline states that the heme is becoming more distorted. 

This is surprising because one would expect for the heme to become more relaxed and less 

distorted due to previously published works [2]. 

References 

PA 21 

⊗ 

A1G A2G B1G B2G 

A1G A1G A2G B1G B2G 

A2G A2G A1G B2G B1G 

B1G B1G B2G A1G A2G 

B2G B2G B1G A2G A1G 

Fig. 1 – The resonance Raman active modes are shown with the 

various deformations along with the respective cross products. 

[1] S. Hu, K. S. Smith, T.G. Spiro. J. Am. Chem. Soc. 118, 12638 (1996). 

[2] A. Hagarman, L. Duitch, R. Schweitzer-Stenner, Biochemistry. 47 (36), 9667-9677.(2008).


Fourier transform infrared spectroscopy to monitor 

embryonic stem cell differentiation 

D. AMI 1 , A. NATALELLO 1 , T. NERI 2 , P. MEREGHETTI 3 , M. ZANONI 2 , M. ZUCCOTTI 4 , S. 

GARAGNA 2 , S. M. DOGLIA 1 AND. C A. REDI 2,5 

1. Dip. di Biotecnologie e Bioscienze, Università di Milano-Bicocca, 

Piazza della Scienza 2, Milan, I-20126, Italy 

2. Lab. di Biologia dello Sviluppo, Dip. di Biologia Animale, Università 

degli Studi di Pavia, Piazza Botta 9, Pavia, I-27100 Italy 

3. Dip. di Chimica, Università di Sassari, Via Vienna 2, Sassari, I-07100, 

Italy 

4. Dip. di Medicina Sperimentale, Sezione di Istologia ed Embriologia, 

Università degli Studi di Parma, Via Volturno 39, Parma, I-43100, Italy 

5. Fondazione IRCCS Policlinico San Matteo Pavia, Italy 

Fourier Transform Infrared (FT-IR) spectroscopy is emerging as a powerful tool in stem cell 

research [1]. In this work we show the application of this technique to the in situ characterization 

of murine embryonic stem (ES) cell differentiation, in order to monitor possible changes in the cell 

macromolecular content [2]. Undifferentiated cells were plated in a leukemia inhibitory factor (LIF) 

free medium to induce spontaneous differentiation. Undifferentiated and differentiating cells at 4, 

7, 9, and 14 days after LIF removing were measured by FT-IR microspectroscopy. Data were 

analyzed by the principal component analysis and subsequent linear discriminant analysis (PCA- 

LDA) that enabled us to segregate ES cell spectra into well separate clusters. Moreover, this 

statistical method allowed us to identify the most significant spectral changes occurring in the 

biomolecule absorption regions. Interestingly, we observed important changes in the protein 

(1700–1600 cm −1) and in the nucleic acid (1050–850 cm −1) absorption regions between days 4 to 7 of 

differentiation, indicating the expression – at this time – of the specific proteins of the new 

phenotype. In particular, the presence of DNA/RNA hybrid bands (954 cm −1 and 899 cm −1) 

suggests that the transcriptional switch of the genome started at this stage of differentiation. These 

infrared results were found to be in agreement with the biochemical characterization of our 

differentiating cells [2], underlying the great potential of FT-IR spectroscopy in stem cell research. 

References 

[1] P. Heraud, M. J. Tobin, Stem Cell Research, In press doi:10.1016/j.scr.2009.04.002 (2009). 

[2] D. Ami, T. Neri, A. Natalello, P. Mereghetti, S. M. Doglia, M. Zanoni, M. Zuccotti, S. Garagna, C. A. Redi, BBA-Mol. 

Cell Research 1783, 98-106 (2008) 

PA 22


Temperature dependence of carotenoid Raman spectra 

A. ANDREEVA 1 , I. APOSTOLOVA 1 AND M. VELITCHKOVA 2 

1. Sofia University, Faculty of Physics, Department of Condensed Matter 

Physics, 5, J. Bourchier blvd., 1164 Sofia, Bulgaria 

2. Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. 

Bonchev str. bl.21, 1113 Sofia, Bulgaria 

Carotenoids are widespread natural molecules, which play several important physiological 

functions. The protection against high-light stress and reactive oxygen species, realized via the 

quenching of electronic excited states of photosentising molecules, quenching of singlet oxygen 

and scavenging of free radicals, is one of the main functions of carotenoids. To understand the 

mechanism of the carotenoid’s photoprotection function it is essential to have a profound 

knowledge of their excited electronic and vibronic states. In the present study we have 

investigated the most power antioxidants: β-carotene and zeaxanthin by means of resonance 

Raman spectroscopy. The aim is to study in detail their Raman spectra in solution as a function of 

temperature and polarizability of the solvent. To measure the spectra in their natural environment 

two solvents have been used: water and pyridine. The latter has been chosen as its polarizability 

(n=1.5092) is close to that of lipid and membrane protein. The temperature dependence of the most 

intensive ν1 band (assigned to C=C bonds in phase stretching vibrations [1,2]) has been obtained in 

the range from 77 K to 295 K at 488 nm and 514.5 nm excitation. It was found that in pyridine the 

band position is blue shifted linearly with temperature for both studied carotenoids, the 

coefficients of the linearity being different. Two possible explanations for the observed shifts are 

considered and compared: the existence of different carotene isomers [2] and two-mode nature of 

ν1 band predicted by quantum mechanical calculations [3] and recently found experimentally in βcarotene 

dissolved in dichloromethane [4]. 

References 

[1] B. Robert, “The electronic structure, stereochemistry and resonance Raman spectroscopy of carotenoids”, in The 

Photochemistry of Carotenoids, eds. H. Frank, A. Young, G. Britton and R. Cogdell, Dordrecht, Kluwer Acad. Publ., 

189 (1999). 

[2] Y. Koyama and R. Fujii, “Cis-trans carotenoids in photosynthesis: configuration, excited state properties and 

physiological functions” in The Photochemistry of Carotenoids, eds. H. Frank, A. Young, G. Britton and R. Cogdell, 

Dordrecht, Kluwer Acad. Publ., 161 (1999). 

[3] M. Schenderlein, M. A. Mroginski, M. Cetin, E. Schlodder, “Low quantum yield electron transfer pathways in PS II” 

in: Photosynthesis. Energy of Sun: 14th International Congress on Photosynthesis Research 2007, edited by J.F. 

Allen et al., Dordrecht, Springer, 191 (2008). 

[4] N. Tschirner, M. Schenderlein, K. Brose, E, Schlodder, M. A. Mroginski, P. Hildebrandt, C. Thomsen, phys. stat. sol. 

(b) 245, 2225-2228 (2008). 

PA 23


Characterization of secondary structure of S-layer 

proteins isolated from lactobacilli strains by Raman 

spectroscopy 

C. ARAUJO-ANDRADE 1 , P. MOBILI 2 , A. LONDERO 2 , C. FRAUSTO-REYES 3 , G. DE ANTONI 2 , H. 

AVILA-DONOSO 1 , E. A. ARAIZA-REYNA 4 , F. RUIZ 4 AND A. GÓMEZ-ZAVAGLIA 2 

1. Unidad Académica de Física de la Universidad Autónoma de 

Zacatecas. Zacatecas, México 

2. Centro de Investigación y Desarrollo en Criotecnología de Alimentos 

(CIDCA), La Plata, Buenos Aires, Argentina 

3. Centro de Investigaciones en Óptica, A.C, Aguascalientes, Ags. 

México 

4. Facultad de Ciencias, UASLP, San Luis Potosí, S.L.P. México 

S-layer structures are macromolecular paracrystalline arrays that completely cover the bacterial 

cell surface [1,2]. They are attached to the underlying cell wall by non-covalent bonds and usually 

may be dissociated and solubilized into protein monomers by chaotropic agents such as guanidine 

hydrochloride and dissociating agents such as 5 M-LiCl. S-layers have been described in several 

species of Lactobacillus, such as L. acidophilus, L. helveticus, L. casei, L. brevis, L. buchneri, L. fermentum, 

L. bulgaricus, L. plantarum, L. kefir and L. parakefir [3,4]. On the basis that several Lactobacillus species 

with an S-layer structure play essential roles in many fermentation processes in food industry, 

silage fermentations and in probiotics for humans and animals, the structural characterization of 

lactobacilli S-layers has become increasingly important [5]. In recent years Raman spectroscopy 

has broadened its applications due to the high content of molecular structure information that it 

provides. In Food Microbiology, it has been used for microorganism characterization and for the 

analysis of proteins [6,7]. As in FTIR spectroscopy, the position of a given band in a Raman 

spectrum depends on the inter and intramolecular interactions, including peptide-bond angles and 

hydrogen-bonding patterns. Therefore, Raman spectroscopy can also be used for protein structural 

analysis. In this work, S-layer proteins extracted from three species of heterofermentative 

lactobacilli isolated from kefir grains (L. kefir, L. parakefir and L. brevis) have been structurally 

characterized. This approach allowed the determination of secondary structure composition (%) of 

S-layer proteins according to the relative areas of the component bands obtained from the peak 

fitting of the amide I and III regions of the raw Raman spectra [8,9]. 

References 

[1] U. B. Sleytr and T. J. Beveridge, Trends Microbiol. 7, 253–260 (1999). 

[2] M. Sára and U.B. Sleytr, J.Bacteriol. 182, 859–868 (2000). 

[3] S. Avall-Jääskeläinen and A. Palva, FEMS Microbiol. Rev. 29, 511-529 (2005). 

[4] P G. Garrote, L. Delfederico, R. Bibiloni, A. Abraham, P. Pérez, L. Semorile and G. De Antoni, J. Dair. Res. 71, 

222–230 (2004). 

[5] M. Kahala, K. Savijoki and A. Palva, J. Bacteriol. 179, 284–286 (1997). 

[6] P. Rosch, M. Schmitt, W. Kiefer and J. Popp, J. Mol. Struc. 661-662, 363-369 (2003). 

[7] A. Torreggiani and G. Fini, J. Raman Spectrosc. 29(3), 229-236 (1998). 

[8] J.T. Pelton and L.R.McLean, Anal. Biochem. 277, 167-176 (2000) 

[9] P. Mobili, A. Londero, T. Roseiro, E. Eusebio, G. De Antoni, R. Fausto and A. Gómez-Zavaglia, Vibrat. Spectrosc. 

2008, doi: 10.1016/j.vibspec.2008.07.016. 

PA 24


Designing noble-metal nanostructures as versatile 

plasmonic probes and sensors for biomedical 

applications 

S. ASTILEAN, M. BAIA, D. MANIU, F. TODERAS, C. FARCAU, M. IOSIN, V. CANPEAN, S. BOCA 

AND M. POTARA 

Babes-Bolyai University, Faculty of Physics and Center for Nanoscience 

and Nanotechnology, Str. M Kogalniceanu 400084 Cluj-Napoca, 

Romania 

Surface plasmon resonances (SPRs) are collective oscillations of free electrons induced by light in 

noble-metal nanostructures. Bioplasmonics is a new emerging branch of biophotonics aiming to 

use the noble-metal nanostructures which support plasmon resonances as versatile, nanoscale 

probes and tools in biomedical research [1]. In recent years, surface plasmon resonances have 

gained considerable interest for sensing, imaging and detection applications via surface-enhanced 

Raman spectroscopy (SERS), surface enhanced IR absorption (SEIRA), metal-enhanced 

fluorescence (MEF) and localized surface plasmon resonances (LSPR). Currently, there is a need 

for inexpensive, flexible and massively parallel methods for the fabrication of plasmonic 

nanostructures. In this presentation we shall report our recent results on the fabrication of 

plasmonic nanostructures by combining many inexpensive methods ranging from colloidal 

lithography to chemical synthesis [2]. As for example, we employ self-assembled nanospheres as 

lithographic masks to deposit metal and generate periodic arrays of nanoparticles or, alternatively, 

we use biopolymer, protein and cell-assisted synthesis procedures to generate a large variety of 

biocompatible gold nanoparticles. To characterize and correlate the plasmonic response of as 

fabricated nanoparticles with their nanometer-scale morphology and topography we combine 

experimental methods (scanning and transmission electron microscopy, atomic force microscopy, 

optical measurements) with computational methods (FDTD, DDA). We have recently developed 

biocompatible SERS substrates for the detection of low-concentration amino-acids and studied the 

direct interaction between gold nanoparticles (rods, prisms, stars-shaped) and some representative 

biomolecules by combining LSPR, MEF and SERS measurements. The as fabricated substrates 

were exploited not only in SERS but also in SEIRA and both FT-SERS and SEIRA spectra of paminothiophenol 

were successfully recorded from the same metallic substrate [3]. We have 

demonstrated the applicability of metallic films perforated with periodic arrays of subwavelength 

nanoholes as dual molecular plasmonic sensors based on LSPR and SERS. Finally we were able to 

register temporal series of SERS spectra at these specific “hot-spot” locations on regular arrays of 

nanoparticles and observed specific behaviors (fluctuating peak intensities and positions) which 

can be assigned to the signature of single-molecule SERS signal. The fabricated SERS and SEIRAactive 

substrates complemented with LSPR and MEF measurements can hold a significant 

potential for novel biomedical sensing and imaging methods. 

References 

[1] Paras N Prasad, Introduction to Biophotonics, Ed. John Wiley & Sons (2003). 

[2] Monica Baia, Simion Astilean, and Traian Iliescu, Raman and SERS investigations of pharmaceuticals, Ed. Springer, 

(2008). 

[3] Monica Baia, Felicia Toderas, Lucian Baia, Dana Maniu and Simion Astilean, ChemPhysChem 10, 7, 1106 – 1111, 

(2009). 

PA 25


Diagnostic analysis of blood samples from women with 

breast cancer by Fourier transform infrared 

spectroscopy 

A. BAGHERI GARMARUDI 1,2 , M.KHANMOHAMMADI 1 AND K.GHASEMI 1 

1. Chemistry Department, Faculty of Science, IKIU, Qazvin, Iran 

2. Department of Chemistry & Polymer Laboratories, Engineering 

Research Institute, Tehran, Iran 

Changes in the cells and tissues, which are subtle and often not readily detectable in the 

histopathological studies, are shown to be well studied, using Fourier transform infrared (FTIR) 

spectroscopy. Application of FTIR spectroscopy to monitor the biochemical changes in living cells 

has gained considerable importance in recent years. It is possible to identify the changes in the 

level of various cellular biochemicals simultaneously, under in vivo and in vitro conditions, as the 

different metabolites absorb the infrared radiation at different wavenumbers [1,2]. Preparation of 

biopsy samples consists of some harmful steps, as the treatment may distort the cell structure and 

immersion of tissues in lipid solvents, such as xylene, dissolves the tissue lipids. On the other 

hand, tissue samples are normally very heterogeneous, comprising several components and 

different cell types. Some other drawbacks are difficult sampling, low speed sample preparation 

and very sensitive keeping conditions. An idea is to replace whole blood sample instead of tissue 

samples in cancer diagnostic studies, as blood is more homogeneous and the sample preparation 

procedure is very simple while blood is studied. It would be also interesting to investigate the 

blood according to its ability to play the role of traveling media [3]. The present study extends this 

idea by focusing on 3 spectral regions in the IR spectrum of the human blood sample, which are 

1600-1720 cm -1, 1480-1600cm -1 and 1180-1300 cm -1, related to amide I, II and III absorbance signals, 

respectively. There are several absorbance bands in these regions due to the different cellular 

proteins. Our studies were aimed to find the statistically confirmed information concerning the 

changes occur in the spectral patterns during the cancer stages, in order to provide reliable data for 

further studies and to propose a method for diagnosis of breast cancer by FTIR spectroscopy of 

blood. Studies showed that A1635/A1658 ratio is decreased while peak area at 1658-1635 cm-1 

spectral region is increased significantly during stages I to IV. Signals in 1539-1550 cm-1 region 

show to be increased from stage I to IV, but A1550/A1539 does not show any change during 

carcinogenesis. Absorbance at 1203-1276 cm-1 spectral region is increased from stage I to stage IV. 

While A1658/A1276 ratio is a little increased during stage I to IV, A1658/A1203 ratio is decreased 

in this range.The obtained FTIR spectra were statistically analyzed to determine differences 

between malignant status and normal status. LDA was applied to discriminate the two classes 

(normal and cancer).Results showed the prediction of 47 samples to be correct according to 

comparing with pathologic reports while 1 normal tissue spectra were wrongly predicted to be 

cancerous. Thus the accuracy was 97.9%. According to misclassification of 1 normal sample in 

cancer class (in totally 48 spectra consisting of 29 cancer and 19 normal cases), sensitivity and 

specificity of the proposed method were determined to be 100 and 94.7%, respectively. 

References 

[1] Fabian H., Lasch P., Boese M. & Haensch W. Journal of Molecular Structure 661, 411–417 (2003). 

[2] Huo X., Wang X.F., Che X. & Huang W.D. Spectrosc Spectral Anal 21, 614–616 (2001). 

[3] Khanmohammadi M., Ansari M.A., Bagheri Garmarudi A., Hassanzadeh G. & Garoosi G. Cancer Invest 25, 397–404 

(2007). 

PA 26


Detecting molecular interactions in molecularly 

imprinted polymers with Raman and surface enhanced 

Raman spectroscopies 

K. KANTAROVICH 1 , I. TSARFATI 2 , L.A. GHEBER 2 , K. HAUPT 3 , AND I. BAR 1 

1. Department of Physics, Ben-Gurion University of the Negev, Beer- 

Sheva 84105, Israel 

2. Department of Biotechnology Engineering, Ben-Gurion University of 

the Negev, Beer-Sheva 84105, Israel 

3. Compiègne University of Technology, UMR CNRS 6022, 60205 

Compiegne, France 

Molecularly imprinted polymers (MIPs), formed by crosslinking a polymer around a template 

molecule, can be used, following their patterning on surfaces and interfacing with optical methods, 

as biochips. MIP droplets were printed using a pipette or a nano fountain pen (NFP) on gold 

coated glass and on surface enhanced Raman scattering (SERS) - active surfaces, enabling 

measurement of Raman and SERS spectra, respectively. The monitored Raman and SERS bands 

could be related to the taken up compound, allowing direct detection of the template in the 

imprinted droplets. The NFP deposition combined with Raman and SERS open the possibility of 

using this approach for monitoring MIP-based sensors on micro-scale. 

PA 27


Natural polyacetylenes studied by Raman spectroscopy 

and DFT calculations 

MALGORZATA BARANSKA, 1 JAN CZ. DOBROWOLSKI, 2,3 MACIEJ ROMAN 1 

1. Faculty of Chemistry, Jagiellonian University, 3 Ingardena Street, 30-060 Krakow, Poland 

2. National Medicines Institute, 30/34 Chełmska Street, 00-725 Warsaw, Poland 

3. Industrial Chemistry Research Institute, 8 Rydygiera Street, 01-793 Warsaw, 

Poland 

Polyacetylenes with conjugated triple bonds are common in a large number of natural products. In 

most cases these secondary metabolites are unique to individual plant species; usually they occur 

in low concentrations and are not essential to plant cell survival. It is assumed that their function 

or importance is mainly related to ecological aspects as they are used for defense against 

predators, parasites, and diseases. They have proven to be important biologically active 

components that can be used as antibacterial, antimicrobial, antifungal, and antiviral agents. 

Moreover, they also exhibit lavricidal activity and cytotoxicity towards a range of cell lines. This 

study is concentrated on the vibrational analysis of polyacetylenes. The vibrational spectra show 

strong and polarized -C≡C- bands in the region of about 2200 cm -1. As has been discussed before 

[1], the number of triple bonds as well as presence of substituents influence the frequency and 

intensity of the polyacetylene -C≡C- stretching modes. Thus, the spectral position of the -C≡C- 

vibrations and pattern of Raman bands usually provide enough information to recognize the type 

of substitution and to support the identification of polyacetylenes. In this work, the structural unit 

R-C≡C-C≡C-R was selected as a model to study the contribution of the substituent effect. To 

understand the influence of electron-donor-acceptor (EDA) properties of the substituent on the 

-C≡C- spectral pattern the R- groups of diverse EDA characteristics, such as: BH2, F, H, Me, OH, 

etc., were considered in the model calculations. Theoretical Raman and IR spectra of the model 

compounds were calculated at the B3LYP/cc-pVDZ and B3LYP/aug-cc-pVDZ levels. For the 

polyacetylenes supposed to be active in plants yet not available in an isolated form, theoretical 

simulation of the vibrational spectra and comparison it with the registered ones seems to be an 

excellent way to confirm or exclude presence of these compounds in the investigated plants. Such 

an approach was applied to analyze polyacetylenes in roots of Coreopsis grandiflora. Raman 

spectrum obtained from the roots shows band at 2194 cm -1. According to Bohlmann [2], this plant 

should contain a monoacetylene with a substitution by a thiophene ring. Theoretical calculations 

allowed to confirm this assumption. For selected polyacetylenes their in situ distribution was 

investigated by using Raman mapping technique. Additionally, the conformational study was 

performed for falcarinol and falcarindiol, polyacetylenes occurring in carrot roots, which can be 

isolated by using various solvents. In order to interpret the experimental spectra of falcarinol the 

B3LYP/aug-cc-pVDZ IR and Raman spectra were calculated for the selected 26 conformers. 

References 

[1] B. Schrader, H. Schulz, M. Baranska, G. N. Andreev, C. Lehner, J. Sawatzki, Spectrochim. Acta A, 61, 1395-1401 

(2005). 

[2] E. Winterfeld, Ferdinand Bohlmann (1921-1991) und sein wissenschafliches Werk, Liebigs Ann. Chem. I-XXXIV, 

1994 

PA 28


Small amines as building blocks of novel Pt(II) based 

anticancer agents: a structural study 

SARA PADRÃO, ANA M. AMADO AND LUÍS A.E. BATISTA DE CARVALHO 

Química-Física Molecular, FCTUC, Universidade de Coimbra, 3004-535 

Coimbra, Portugal 

The discovery of cisplatin’s (cis-diamminedichloroplatinum (II), cDDP) biological activity by 

Rosenberg opened the door to the use of inorganic agents as chemotherapeutic drugs. However, in 

an attempt to overcome cDDP severe side effects and/or to expand its range of activity, the 

development and biological evaluation of new related metal-based agents that might overcome 

those limitations is nowadays a very active research area. An extensive number of platinum(II) 

complexes are being studied as promising antineoplastic drugs. The chemical characterization of 

both the isolated ligands and the Pt-complex is of crucial importance in this context. On the other 

hand, quantum inorganic chemistry has proven to be a very helpful tool for the interpretation of 

the experimental spectra. A group of platinum complexes that has deserved particular interest in 

the last few years involves different amine ligands. For instance, in vitro studies for the Pt-complex 

comprising one isopropylamine (iPram) and one pyrazole (Py) ligands, in a trans configuration, 

has yielded very promising results in overcoming cDDP resistance [1]. In the present work, a full 

conformational analysis, at the mPW1PW/6-31G* quantum theory level, is performed for both 

these ligands. A complete assignment of the experimental vibrational spectra (Raman and FTIR) is 

carried out, in the light of the theoretical prediction of the spectra. In order to assess the 

importance of the hydrogen bonding network on the molecular structure and on the vibrational 

profile of these chelates, solutions of the ligands in both water and CCl4 (which does not allow the 

formation of hydrogen bonds) are also considered, both experimental and theoretically. Finally, 

the structural preferences of the corresponding t-complexes are evaluated, by mean of theoretical 

calculations. 

References 

[1] E. Pantoja, A. Gallipoli, S. Van Zutphen, S. Komeda, D. Reddy, D. Jaganyi, M. Lutz, D.M. Tooke, A.L. Spek, C. 

Navarro-Ranninger, J. Reedijk, Journal of Inorganic Biochemistry 100, 1955-1964 (2006). 

PA 29


An advanced biosensing SERS-based method 

F. DOMENICI , A. R. BIZZARRI, AND S. CANNISTRARO 

Biophysics and Nanoscience Centre (BNC), Università della Tuscia and 

CNISM, Largo dell’Università, 01100 Viterbo (Italy) 

One of the main challenge in bio-medicine is the improvement of the detection sensitivity to 

achieve tumor-marker recognition at very low concentration, when the disease is not yet 

significantly advanced [1]. In the last years, nano-approaches and single molecule spectroscopies, 

provided new tools to the biorecognition-based traditional methods, in order to optimize the 

ultrasensitivity analytical detection. Surface Enhanced Raman Scattering (SERS) technique has 

huge potential for the development of sensitive and quantitative analytical methods [2,3]. We 

propose an advanced biosensing SERS approach aimed at the synergic combination of both the use 

of nanostructured materials and biorecognition process to increase the sensitivity of the analytical 

assay. By exploiting the high increment of the Raman signal-to-noise ratio in SERS, we are able 

to reveal the presence of tumor-markers below the pico-molar concentration. We assessed the 

effectiveness of the method, focusing on the interaction between p53 and Azurin (Az). p53, which 

has a pivotal role in cancer defense mechanisms, was demonstrated to be stabilized by the 

interaction with the redox protein Az [4]. We assembled p53 on 50 nm gold nanoparticles (Np) 

by means of a SERS active bifunctional linker (4-ATP) (see Fig. 1A) and subsequently we dropped 

the obtained nanoparticle-protein hybrid system on a capture biosensing platform composed of Az 

monolayer to allow the p53 recognition (see Fig. 1B). By following the SERS signal of 4-ATP-Np 

marker, we detected a threshold limit concentration of 10 -13M of p53 captured by the 

functionalized Azurin substrate. The SERS-based approach was also supported by Atomic 

Force Microscopy (AFM) imaging, also in the perspective to lower the threshold sensitivity. 

Fig. 1 – A) p53 labeled with 4-aminothiophenol (4-ATP)-Np SERS marker; B) p53-Az biorecognition 

event. 

References 

[1] N. L. Rosi and C. A. Mirkin. Chemical Reviews 105 (2005) 1547. 

[2] A. Otto, I. Mrozek, H. Grabhorn, and Y. Pommier, J. Phys.: Condens. Matter 4 (1992) 1143. 

[3] A. R. Bizzarri, S. Cannistraro, Nanomedicine 3 (2007) 306. 

[4] M. Taranta, A. R. Bizzarri and S. Cannistraro, J. Mol. Recognit. 21 (2008) 63. 

PA 30


Raman and multivariate statistical methods applied to 

human blood platelets 

C. W. BLACKLEDGE 1 , E. REDDY 2 , S. O'NEILL 2 , N. MORAN 2 , R. J. FORSTER 1 AND T. E. KEYES 1 

1. National Biophotonics and Imaging Platform, Dublin City University, 

Glasnevin, Dublin 9, Ireland 

2. Clinical Research Centre and Clinical Pharmacology, Royal College of 

Surgeons in Ireland, Dublin, Ireland 

Many commercial advances in vibrational spectroscopy instrumentation and statistical analysis 

software have been made in recent years. With the aid of statistical methods, Raman spectroscopy 

has been used to discriminate healthy and diseased tissue, and can provide an alternative 

diagnostic methodology compared to lengthy experimental protocols and sometimes subjective 

image interpretation carried out by trained and experienced pathologists. Relying on the intrinsic 

vibrational transitions of molecules, Raman has great potential as a minimally invasive technique 

for characterization as well as microscopic and sub-microscopic imaging. We present the use of 

multivariate statistics on the Raman spectroscopy of human blood platelets. As is the case for 

many complex biological materials, platelets consist of many different compounds such as lipids, 

proteins, and carotenoids, and this complicates the direct interpretation of vibrational spectra. 

Principle component analysis of the fingerprint region between 400 cm -1 and 1700 cm -1 aids in 

simplifying the interpretation of large sets of Raman spectra by projecting the data set into a 

smaller dimensional space. For example, 1024 channels of Raman spectral intensity measurements 

are reduced to three principal components. Previous work in our group showed that multivariate 

statistical analysis of Raman spectra of blood platelets could be used to discriminate between 

platelets treated with different agonists. More recently we have begun to apply similar statistical 

techniques to uncover the possibility of examining chemical and structural changes that may be 

observed with Raman spectroscopy of cellular molecular species in living human platelet cells. We 

will explain our recent results in view of the many opportunities for the application of Raman and 

statistical methods in biological and medical problems. 

PA 31


Study of humane bone tissue regeneration by Raman 

micro-spectroscopy 

Z. BLASZCZAK 1 , T. BUCHWALD 2 AND M. KOZIELSKI 2 

1. Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61- 

614 Poznań, Poland 

2. Faculty of Technical Physics, Poznan University of Technology, 

Nieszawska 13A, 60-965 Poznań, Poland 

Biomechanical properties of human bones are determined, besides the mass, by the chemical 

composition of bones (types of minerals and the ratio of their content to the content of collagen) 

their structural properties (tissue organisation, orientation and curing of collagen). The method of 

Raman micro-spectroscopy permits identification of the bone tissue components, detection of 

surface changes in bone tissue and orientation of collagen fibres. This information permits 

evaluation of the mechanical resistance of the bones and their susceptibility to break. Changes in 

the structure of the bone tissue (composition and arrangement of the collagen fibres) were studied 

in the thigh bone of man by Raman micro-spectroscopy. Some bands in the Raman spectrum of the 

bone tissue were found to be sensitive to the polarisation of the excitation beam and the scattered 

beam. The intensity of these bands changed according to the orientation of the polarisation vector 

of these beams. The bands less sensitive to the polarisation of the excitation and scattered beams 

provide information on changes in the chemical composition of the bone tissue, while the bands 

more sensitive to the polarisation permit concluding on the orientation of the organic component 

in the bone tissue. The information can be used for evaluation of the bone tissue regeneration. 

References 

[1] G. Penela, C. Delfossea, M. Descampsb, G. Leroya, Bone 36, 893 – 901, 2005. 

[2] M. Kazanci, H.D. Wagner b, N.I. Manjubala, H.S. Gupta , E. Paschalis, P. Roschger, P. Fratzl, Bone 41, 456– 461, 

2007 

[3] M. Kazanci, P. Roschger, E.P. Paschalis, K. Klaushofer, P. Fratzl, Journal of Structural Biology156, 489–496, 2006. 

PA 32


Trp - modified bovine purine nucleoside phosphorylase 

K. BREER 1 , B. WIELGUS-KUTROWSKA 1 , A. GIRSTUN 2 , K. STAROŃ 2 AND A. BZOWSKA 1 

1. Dept. of Biophysics, Institute of Experimental Physics, University of 

Warsaw, świrki i Wigury 93, 02-089 Warsaw, Poland 

2. Dept. of Molecular Biology, Institute of Biochemistry, University of 

Warsaw, Miecznikowa 1, 02-089 Warsaw, Poland 

Purine nucleoside phosphorylase (PNP) leads the reversible phosphorolytic cleavage of the 

glycosidic bond of purine nucleosides and some analogues in the presence of phosphate. PNP is of 

interest as a drug target because of its role in some immunological diseases and in the intracellular 

degradation of some anti-tumor and anti-viral drugs [1]. The enzyme is a trimer with three 

tryptophan residues in every subunit. However, these tryptophan residues are remote from the 

PNP active site and convenient determination of parameters of binding by means of fluorescence is 

limited only to ligands which exhibit they own fluorescent signal when bound to the enzyme [2]. 

By site-directed mutagenesis we introduced additional Trp residue in the bovine PNP sequence in 

a few positions in the active site neighborhood and the regions 32-36, 56-69, 244-266 reported to 

undergo a conformational change upon ligand binding [3]. We modified only aromatic aminoacids 

or non conserved positions, which were 35, 36, 56, 70, 159, 200, 249, 262 and 266. All the mutants 

exhibit similar to the wild-type enzyme properties of binding of guanine and activity with inosine, 

but only two of them Phe159Trp (similarly to human PNP mutant [4]) and Phe200Trp show 

exeptional fluorescent properties. These mutants respond with a unique strong signal change due 

to ligand binding and were used for determination of dissociation constants, rates and 

stoichiometry of binding of multisubstrate analogues DFPP-G and (±)-cis-piranyl [5] (Fig. 1). 

References 

Fig. 1 Phe200Trp bovine purine nucleoside phosphorylase mutant 

responds with about a 4 times stronger fluorescence signal 

quenching upon (±)-cis-piranyl binding (squares) when compared 

to the wild-type enzyme (open circles). 

[1] A. Bzowska, E. Kulikowska, D. Shugar, Pharmacol. Ther. 88, 349-425 (2000) 

[2] D. J. Porter, J. Biol. Chem. 267, 7342-7351 (1992) 

[3] C. Mao, W. J. Cook, M. Zhou, A. A. Fedorov, S.C. Almo, S.E. Ealick, Biochemistry 37, 7135-7146 (1998) 

[4] M. Ghanem, N. Zhadin, R. Calleder, V.L. Schramm, Biochemistry 48, 3658-3668 (2009) 

[5] L. Glovaš-Obrovac, M. Suver, S. Hikishima, T. Yokomatsu, A. Bzowska, Nucleosides Nucleotides Nucleic Acids 26, 

989-993 (2007) 

PA 33


Metal binding motifs of adenylate kinase and ATP 

sulfurylase 

S.A. BURSAKOV 1 , A. V. KLADOVA 2 , O. YU. GAVEL 1,2 , V.L. SHNYROV 2 , C.BRONDINO 3 , I. 

MOURA 2 , J. J.G. MOURA 2 

1. Departamento de Protección Ambiental, Estación Experimental del 

Zaidin (EEZ-CSIC), 18008 Granada, Spain (sergey.bursakov@eez.csic.es) 

2. REQUIMTE, Departamento de Química, Centro de Química Fina e 

Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova 

de Lisboa, 2829-516 Caparica, Portugal 

3. Facultad de Bioqumica y Ciencias Biologicas, Universidad Nacional 

del Litoral, 3000ZAA Santa Fe, Argentina 

Three different metal ions zinc, cobalt and iron were found to be present in adenylate kinase (AK) 

from few Gram-negative bacteria where the structural motif Cys-X2÷5-Cys/His-X12-16-Cys-X2-Cys in 

the LID domain of AK is responsible for their binding [1]. Similar but different motif Cys-X2-Cys- 

X8-Cys-X-His containing cobalt or zinc was found in ATP sulfurylase (ATPS) from Desulfovibrio 

desulfuricans ATCC 27774 and Desulfovibrio gigas [2]. In this work, we isolated recombinant 

homogeneous Co 2+-AK, Zn 2+-AK and Fe 2+-AK of D. gigas and native ATPS from D. desulfuricans. 

Their conformational stability, spectroscopic and kinetic properties were studied. Binding sites of 

cobalt holo forms of AK and ATPS were compared by EPR saturation studies. The comparison 

indicates that the Co 2+ ions have different relaxation properties. Tertiary structures of holo-AK 

were compared using near-UV circular dichroism analysis. The near-UV CD of proteins arises 

from the environment of aromatic amino acids side chains. The contributions of phenylalanine and 

tryptophan residues in all holo-AK forms are similar, when the contribution of tyrosine residues is 

very different. The calorimetric behavior of holo-forms is also different. Thus, reversible 

denaturation process was detected for Co- and Zn- forms at pH 10, while Fe-AK denaturation is 

irreversible at broad pH range. 

References 

[1] O.Yu. Gavel, S.A. Bursakov, G. Di Rocco, J. Trincão, I.J. Pickering, G.N. Graham, J.J. Calvete, V.L. Shnyrov, C.D. 

Brondino, A.S. Pereira, J. Lampreia, P. Tavares, K.J.G. Moura, I. Moura JIB 102, 1380-1395 (2008) 

[2] O.Yu. Gavel, S.A. Bursakov, J.J. Calvete, G.N. George, J.J. Moura, I. Moura. Biochemistry 37, 16225-16232 

(1998) 

PA 34


Detection of pesticide damage in human keratinocytes 

by means of Raman microspectroscopy 

G. PERNA 1 , M. LASALVIA 2 , A. CASTRO 1 , N. L’ABBATE 2 , E. MEZZENGA 1 , G. QUARTUCCI 1 AND 

V. CAPOZZI 1 

1. Dipartimento di Scienze Biomediche, Università di Foggia, 

Viale Pinto, I-71100 Foggia, Italy 

2. Dipartimento di Scienze Mediche e del Lavoro, Università di Foggia, 

Viale Pinto, I-71100 Foggia, Italy 

Raman microspectroscopy has been used to detect biochemical and structural changes in cultured 

human keratinocytes as a consequence of chlorpyriphos exposure. Chlorpyriphos is an important 

member of the organophosphate pesticides: in fact, it exhibits insecticidal activity against a wide 

range of insects on foliage in a wide range of crops. It damages mainly the central nervous system 

and also the cardiovascular and respiratory ones. So, it is present in a large number of commercial 

products. Some moderately toxic effects are observed in humans when it is used together with 

other pesticides. Only in exposed workers some chronic effects have been evidenced, as loss of 

memory and concentration, confusion, headache and insomnia. Therefore, the study of the effetcs 

of chlorpyriphos exposure is important to protect humans, in particular those people employed in 

the manufacturing and utilization activities and consumers. Studies of exposed cultured cells are 

essential for the investigation of the biological response to pesticides at cellular level, in order to 

evaluate eventual biochemical modifications caused by such products. Keratinocytes cultured cells 

have been treated with increasing doses of chlorpyriphos for 24h and concentrations from 10 -3 M to 

10 -6 M. A viability test, performed by means of the Trypan blue assay, indicated that exposure to 

10 -3 M of chlorphyriphos for 24 h can be considered a cytotoxic dose. Both control and treated cells 

have been analysed by means of Raman microspectroscopy. We have found that important 

structural and biochemical changes occur in treated keratinocytes. In particular, such 

modifications, related to the structure of DNA bases, single aminoacids and proteineous linkage 

between aminoacids, begin after exposure at chlorphyriphos solution at the lowest concentration 

(10 -6 M). Therefore, cellular damage starts after exposure to chlorpyriphos doses well below that 

established as cytotoxic. 

PA 35


Intramolecular and Intermolecular Charge Transfer Spectroscopy in Different 

Environment including some heterogeneous medium of nano size 

P. CHOWDHURY 

Department of Physics & Material Science, Jaypee Institute of Information 

Technology University, A-10, Sector-62, Noida-201307,UP, India 

This is an effort to explain the ground and excited state intra/intermolecular charge transfer 

photophysics and photochemistry of five member heterocyclic compounds like Pyrrole-2- 

Carboxyldehyde (PCL), Pyrrole-2-carboxylic acid (PCA) in different environment and in some 

heterogeneous nano cavity like cyclodextrins and micelles. Existence of charge transfer complex in 

different environment have been successfully explained with ab initio DFT (time dependent), HF, 

CIS, PM3, ONIOM calculations. The experimental and theoretical data shows the existence of 

intramolecular hydrogen bonding between H6….O11 of pyrrole and formyl group in ground state 

and possibility of intramolecular and intermolecular proton transfer from pyrrole group(-NH) to 

formyl group (-C=O) in the excited state. The effect of confinement of guest molecule inside the 

cavity of cyclodextrin has been studied. The mechanism of diffusion and surface tension are used 

to analyze the host guest interaction and was demonstrated by measuring the binding force and 

dipole moment of cyclodextrin-guest molecule in liquid environment at the molecular level. The 

specific strong interaction of water molecule was confirmed on the nano scale. Quantum chemical 

calculations on the charge density distribution and stretching of N-H bond indicated that PCA, 

PCL are good candidate for intramolecular proton transfer in excited state [1,4]. Possibility of 

intramolecular and intermolecular hydrogen bonding of PCL in ground state was established 

theoretically by the distance N5-H6………O11 of acidic and basic moieties of PCL and 

experimentally it is verified by the IR stretching and bending mode vibrations of different parts of 

the molecule. The possibility of transfer of hydrogen from pyrrole ring towards [……………] 

Absorbance 

0.20 

0.15 

0.10 

0.05 

0.00 

200 250 300 350 

Wavelength (nm) 

PCL in Methanol 

PA 36 

IR Absorbance (arb.unit) 

1654 

1664 

1602 

1500 1000 500 

1446 

1404 

1356 

1314 

1450 

1478 

1341 

1400 

Experimental (b) 

1140 

1094 

1048 

Theoretical (a) 

1166 

1127 

1089 

996 

960 

920 

864 

862 

792 

754 

778 

715 

606 

629 

518 

513 

1500 1000 500 

Wavenumber (cm-1) 

Fig. 1 – UV-VIS absorption spectra of PCL in MeOH. IR absorption spectra of PCL in KBr pellets. 

References 

1. P.Chowdhury, S.Panja, A. Chatterjee, P. Bhattacharya, S. Chakravorti, J. Photochem. Photobiol A: Chem. 170, 

(2004), 131. 

2. P.T.Chou, W.S.Yu, Y.C.Chen, C.Y.Wei, S.S.Martinez, J Am Chem Soc, 120, (1998), 12927. 

3. F.Jensen, Introduction to Computational Chemistry, Wiley, England, (1999). 

4. P.Chowdhury, S.Panja, S.Chakravorti, J Phys Chem A, 107, (2003), 83.


Influence of level of hydration on vibrational dynamics 

of Dipalmitoyl Phosphatidylcholine 

P. CIACKA 1 AND H. ABRAMCZYK 1,2 

1. Laboratory of Laser Molecular Spectroscopy, Institute of Applied 

Radiation Chemistry, Technical University of Lodz, Ul. Wroblewskiego 

15, 93-590 Lodz, Poland 

2. Max-Born Institute, Max-Born Str. 2A, 12489 Berlin, Germany 

Our goal is to investigate if water molecules bound with H-bonds to Dipalmitoyl 

Phosphatidylcholine (DPPC) phosphate and carbonyl groups affect vibrational dynamics of CH 

groups in the molecule’s alkyl chain. Results we have obtained by using time resolved infra-red 

pump-probe spectroscopy show that the amount of bound water influences the timescales of 

vibrational dynamics even in the groups far away from where water lies, which is the case 

especially for CH3 group. We also observe spectral diffusion of water band occurring before 

vibrational relaxation. Fast times of this process lead us to believe that it is related to energy 

transfer between water molecules involved in hydrogen bonds. Two-color experiment, where 

pump pulse was set to the OH region of water and probe to the CH region of DPPC, has revealed a 

feature which depended on water content of the sample. We have assigned it to energy transfer 

between water and lipid. The experiment performed with pump tuned further to the blue showed 

no signal for lipids. Based on that, we could deduce that the signal depends on how close (or how 

strongly bound) water is to the binding site in the lipid. 

PA 37


Towards a better knowledge of fluorescence properties 

of humic acid using fractionation 

C. COELHO 1 , G. GUYOT 1 , A. TER HALLE 1 , C. RICHARD 1 , O. TRUBETSKOJ 2 AND O. TRUBETSKAYA 3 

1. Laboratoire de Photochimie Moléculaire et Macromoléculaire, UMR 

n°6505 CNRS - Université Blaise Pascal 63177, Aubière Cedex, France 

2. Institute of Basic Biological Problems, Russian Academy of Sciences, 

142292, Pushchino, Moscow region, Russia 

3. Branch of Shemyakin and Ovchinnikov Insitute of Bioorganic 

Chemistry, Russian Academy of Sciences, 142292, Pushchino, Moscow 

region, Russia 

Humic acid from a chernozem soil (Kursk region, Russia) has been fractionated by coupling size 

exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE) on three 

fractions A, B and C+D. The bulk humic acid and their fractions present fluorescence and 

photoinductive properties that were compared in the past [1]. It was demonstrated that most of 

fluorophores and a great part of the photoinductive chromophores are located in the lowest 

molecular size fractions of soil humic acid (C+D) [2]. As shown in the figure 1, two types of 

fluorophores are present in the bulk humic acid those emitting at 510 nm and those emitting at 550 

nm. Fractions A and B have lost their emitting properties : there’s a residual fluorescence of 

trypophane-like structures at 350 nm for a 280 nm excitation. Fraction C+D fluorescence is located 

550 nm and is four times more intense than the bulk humic acid. Using SEC-PAGE, we lost the 

emission at 510 nm from the bulk humic acid. Recently a new fractionation based on ultrafiltration 

enables to prepare quantitative low molecular size fractions of humic acid : ultrafiltrates below 5 

kDa. It was shown that they contain C+D fractions [3]. These ultrafiltrates below 5 kDa present the 

emission at 510 nm and are much more photoactive than C+D fractions. Combining optical 

properties and fractionation, we hope to better characterize the type and the nature of 

chromophores which are involved in humic acids. 

References 

PA 38 

Fig. 1 – Emission and Excitation matrix of Kursk 

humic acid and its fractions A, B and C+D obtained by 

SEC-PAGE, emission intensity is normalised to a 

dissolved organic carbon of 1 mol.L -1 

[1] J.P. Aguer, O. Trubetskaya, O. Trubetskoj, C. Richard, Chemosphere 44, 205-209 (2001) 

[2] C. Richard, O. Trubetskaya, O. Trubetskoj, O. Reznikova, G. Afanas'eva, J.P. Aguer, G. Guyot, Environmental 

Science & Technology 38, 2052-2057 (2004) 

[3] O. Trubetskoj, O. Trubetskaya, G. Afanas'eva, O. Reznikova, C. Saiz-Jimenez, Journal of Chromatography A 767, 

285-292 (1997)


Ultrafast photochemistry in protochlorophyllide by 

femtosecond infrared spectroscopy 

M. COLINDRES 1 , R. GROß 1 , S. SEIDEL 2 , G. HERMANN 2 AND R. DILLER 1 

1. Fachbereich Physik, TU Kaiserslautern, Kaiserslautern, Germany 

2. Institut für Biochemie und Biophysik, Universität Jena, Jena, Germany 

The light dependent reduction of protochlorophyllide (PChlide), the precursor in the biosynthesis 

of chlorophyll, is catalyzed by the enzyme NADPH:protochlorophyllide oxidoreductase (POR). 

Excitation of PChlide initiates a series of processes on the excited electronic state energy surface in 

the femto-nanosecond time regime [1]. To further characterize the photochemistry of PChlide, 

specifically its excited state dynamics, we have studied the primary photoreaction of PChlide in 

methanol by means of sub-picosecond mid-infrared spectroscopy. Preliminary results (Fig. 1) in 

the carbonyl region indicate changes of a C-O stretch vibration upon electronic excitation. 

Systematic measurements in a broad spectral region, including C-O and C-C stretch vibrations will 

be presented and discussed in the context of the PChlide photochemistry. 

References 

Fig. 1 – Absorption kinetics at 1724 cm -1 after excitation of PChlide 

at 630 nm 

[1] H. B. Dietzek, S. Tschierlei, G. Hermann, A. Yartsev, T. Pascher, V. Sundström, M. Schmitt and J. Popp, 

ChemPhysChem 10, 144-150 (2009). 

PA 39


Some more facts on the molten globule state 

B. CZARNIK-MATUSEWICZ 1 , S. R. RYU 2 AND Y.M. JUNG 2 

1. Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland 

2. Department of Chemistry, Kangwon National University, Chunchon, 200-701, Korea 

The molten globule state is not an independent conformational state, but an equilibrium mixture of 

the unfolded and native states that was confirmed by X-ray scattering [1].The molten globule 

generally defined as a state with substantial secondary structure, absence of native-like tertiary 

structure, compact and globular is not describe at the same levels of details as the two limited 

states. We have employed FTIR-ATR and CD spectroscopies to studies the structural features of 

the molten globule of Ca 2+-bond (holo) α-lactalbumin. The infrared experiment was done in a 

function of pH and temperature for the aqueous solutions and the thin film samples. 

Measurements for pH from 8 to 2 allowed us to analyze the conformational changes 

accompanying the transition from the native to the molten globule state. The CD Far-UV and 

Near-UV data disclosed fact that changes at the level of the ternary and the secondary structure 

cannot be described as a strictly correlated process developed according to the same pH-profile. A 

secondary structure evolution towards non-native helical configuration precedes decay of the 

ternary arrangement. This fact was confirmed by the CD and the FT-IR data independently 

analyzed by means of the two-dimensional correlation spectroscopy (2DCOS) and the principal 

components analysis (PCA) combined with the multivariate curve resolution-alternating least 

squares (MCR-ALS), approaches that are very efficient in analysis of infrared spectra of 

biomolecules studied in aqueous environment [2]. The three resolution methods enabled to detect 

an intermediate state before the molten globule is achieved at pH 2. Analysis of the native, the premolten, 

and the molten globule reveals that both levels of structure, i.e. the secondary and the 

ternary are modified. Temperature-dependent studies done for the native (pH 8) and the molten 

globule (pH 2) structure let us characterize thermal stability of the two states. Obtained results 

point on a larger structural evolution of the native than the molten globule form during heating 

process from 25 oC to 90 oC. 

References 

1750 1700 1650 1600 1550 1500 1450 1400 

wavenumber (cm-1 1750 1700 1650 1600 1550 1500 1450 1400 

wavenumber (cm ) -1 ) 

PA 40 

pH 

2 

3 

3.5 

4 

4.5 

5 

6 

7 

8 

Fig. 1 – FTIR-ATR spectra of (holo) 

α-lactalbumin measured as a thin film 

on a diamond crystal in function of pH. 

[1] M. Kataoka, K. Kuwajima, F. Tokunaga, Y. Goto, Protein Scien. 6, 422-430 (1997). 

[2] B. Czarnik-Matusewicz, S.B. Kim, Y.M. Jung, J. Phys. Chem. B, 113, 559–566 (2009).


Preferential solvatation of TFE in lysozyme: a 

luminescence study 

M. D'AMICO 1 , M. CANNAS 2 , V. MARTORANA 1 , S. RACCOSTA 1,2 , AND M. MANNO 1 

1. Inst. of Biophysics, Italian National Council of Research, Via Ugo La 

Malfa 153, Palermo, I-90146, Italy 

2. Dept. of Physical and Astronomical Sciences, University of Palermo, 

Via Archirafi 36, Palermo, I-90123, Italy 

The effect of alcohols on the conformational and thermodynamic stability of proteins in solution is 

an important issue for the understanding of the physics of the solubility, misfolding and 

aggregation of proteins. Here, we study the influence of the trifluoroethanol (TFE), on the 

thermodynamic stability of a model protein, the hen egg-white lysozyme, by stationary and time 

resolved photoluminescence (PL) of its tryptophans (TRP). TFE is known to induce conformational 

changes in proteins, either by a “macroscopic” solvent-mediated effect or by a direct interaction 

with the protein [1]. In Fig. 1 we show that the first moment of stationary PL of TRP shows a 

sigmoid red-shift for each TFE concentration, compatible with a thermal denaturation process. The 

characteristic temperature decreases for high TFE content accordingly with calorimetric data [2]. 

At a fixed temperature the TFE gives a blue-shift of the TRP band which is more pronounced at 10 

(~4 nm) than at 80 °C (~1 nm) [3]. The time resolved PL excited by laser shows a “dynamic” red 

shift of the first moment, during the decay in the nanosecond range, which is almost independent 

on the TFE concentration. All these results point towards a microscopic picture where the TFE 

forms a preferential solvation shell around the protein [4], thus decreasing the thermal stability of 

the protein. 

References 

Fig. 1 –First moment of Lysozyme TRP band as a function of 

temperature and TFE concentration. The lines represent the fit with 

the reported function. 

[1] R. Walgers, T. C. Lee, A. Cammers-Goodwin, J. Am. Chem. Soc. 120, 5073-5079 (1998). 

[2] V. Bhakuni, Arch. Biochem. Biophys. 357, 274-284 (1998). 

[3] J. F. Povey, C. M. Smales, S. J. Hassard, M. J. Howard, J. Struct. Biol. 157, 329-338 (2007). 

[4] R. Carrotta, M. Manno, F. M. Giordano, A. Longo, G. Portale, V. Martorana, P. L. San Biagio, Phys. Chem. Chem 

Phys. DOI: 10.1039/b818687a (2009). 

PA 41


Fluorescence study of the interaction between 

antitumoral drug emodin and bovine serum albumin in 

presence of silver nanoparticles 

R. DE LLANOS 1 , P. SEVILLA 1, 2 , C. DOMINGO 1 , S. SÁNCHEZ-CORTÉS 1 AND J.V. GARCÍA-RAMOS 1 

1. Instituto de Estructura de la Materia, Consejo Superior de 

Investigaciones Científicas, Serrano 121, Madrid, 28006, Spain. 

2. Departamento de Química Física II, Facultad de Farmacia, 

Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 

28040, Spain. 

Emodin has been known for a long time for its laxative and anti-inflammatory effects. Recent 

studies have shown that it can induce apoptosis in several cancer cells [1]. This orange crystalline 

compound belongs to anthraquinone family and it is one of the active components of rhubarb. 

Emodin is able to form complexes with some proteins like bovine serum albumin (BSA) which 

ensure drug delivery through plasma. Nowadays, metal nanoparticles are being used to release 

drugs in a controlled and targeted way. Thus, the behaviour of emodin-BSA complex in presence 

of metal nanoparticles has great importance in the design of new drug delivery systems. In this 

work we study the binding of emodin to bovine serum albumin adsorbed on silver colloids. We 

have used different spectroscopic techniques like UV, fluorescence (frequency domain and time 

domain) and Circular Dichroism. It has been shown that the secondary structure of the protein is 

modified by the absorption on nanoparticles. The analysis of the binding saturation curve has 

allowed us to determine only a single binding site for emodin. This differs from previous results 

obtained for the complex in aqueous solution and in the absence of Ag nanoparticles [2] where the 

binding is produced at the two Sudlow´s sites of the protein. Furthermore, fluorescence lifetime 

studies have been carried out for the protein-emodin complex, in absence and presence of 

nanoparticles. Results indicate decrease in protein fluorescence lifetime when nanoparticles are 

present and also when drug concentration increases. 

References 

Fig. 1 – Structure, numbering and acid-base equilibrium of emodin 

[1] Z. W. Huang, G. C. Chen, P. Shi, Cancer Detection and Prevention 32, 286-291 (2009). 

[2] P. Sevilla, J. M. Rivas, F. García-Blanco, J. V. García-Ramos, S. Sánchez-Cortés, Biochimica Et Biophysica Acta 

Proteins and Proteomics 1774, 1359-1369 (2007). 

PA 42


Laser Raman Micro-spectroscopy of Proterozoic 

and Palaeozoic organic walled microfossils 

M.C DHAMELINCOURT 1 , A. MEZZETTI 1 , GERARD VERSTEEGH 2 , MARCO VECOLI 3 

1. Laboratoire de Spectrochimie Infrarouge et Raman UMR CNRS 8516, 

Bat C5, USTL, Cité Scientifique, 59655 Villenenuve d’Ascq, France 

2. Organic Geochemistry Department 

Center for Marine Environmental Sciences, MARUM 

University of Bremen, Bremen, Germany. 

3. Laboratoire de Paléontologie UMR CNRS 8157 Geosystems, USTL, 

Cité Scientifique, 59655 Villenenuve d’Ascq, France 

Proterozoic and Palaeozoic organic walled microfossils (palynomorphs) comprise a wide range of 

organisms (including oceanic photosynthetic microplancton, microzooplankton, and microscopic 

spores from earliest land plants), which record fundamental evolutionary events in Earth’s 

biosphere. Classical comparative morphology analyses can be successfully applied only in a few 

cases when directly measurable morphological features are available but in the more general cases 

the biological affinity of Proterozoic and Early Palaeozoic palynomorphs remain largely unknown. 

Recently, new techniques based on microchemical analysis of individual organic-walled 

microfossils demonstrated their potential for elucidating the cellular anatomy, composition, and 

mode of preservation of microfossils, thus offering new insights into the palaeobiology of ancient 

microorganisms. In the last few years, Laser Raman micro-spectroscopy has emerged as one of the 

most powerful analytical tools (see for instance [1, 2]). In this study, different kinds of 

exceptionally well preserved palynomorphs extracted from Precambrian to early Devonian 

sediments were individually analyzed by Laser microRaman spectroscopy technique in order to 

better characterize their chemical composition. The results will be discussed in the framework of 

the current knowledge on organic-walled microfossils. Possible improvements of the microRaman 

analysis, either by new instrumentation / new data treatment techniques (e.g. chemometrics) or by 

the synergic use of complementary techniques (e.g. micro-FTIR) will be also discussed. 

References 

[1] S. Bernard, O. Beyssac, K. Benzerara, Appl. Spectrosc. 62, 1180-1188 (2008) 

[2] J.W. Schopf, A.B. Kudryavtsev, D.G. Agresti, A.D. Czaja, T.J. Wdowiak, Astrobiology 5, 333-371 (2005) 

PA 43


L-isoserine conformation in low-temperature IR matrix 

spectra interpreted by means of the DFT calculations 

J.CZ. DOBROWOLSKI 1,2 , M.H. JAMRÓZ 2 , R. KOŁOS 3 , J.E. RODE 2 , J. SADLEJ 1,4 

1. National Medicines Institute, 30/34 Chełmska Street, 00-725 Warsaw, Poland 

2. Industrial Chemistry Research Institute, 8 Rydygiera Street, 01-793 Warsaw, Poland 

3. Institute of Physical Chemistry, PAN, 44 Kasprzaka Street, 01-224 Warsaw, Poland 

4. Faculty of Chemistry, Warsaw University, 1 Pasteura Street, 02-093 Warsaw, Poland 

Izoserine, NH2CH2CH(OH)COOH, 3-amino-2-hydroxypropionic acid (Scheme 1), is serine isomer 

in which the NH2 and OH groups were interchanged. Isoserine is the β-amino acid - an amino acid 

with the NH2 placed in the β position. A few β-amino acids have been isolated from plants, 

bacteria, and invertebrates. Paclitaxel (Taxol), first isolated from the bark of Taxus brevifolia L, is the 

most important natural compound composed of the isoserine molecule, which is used in 

chemotherapy of breast, ovarian, and lung cancers. The presence of the additional carbon atom in 

the backbone of a β-amino acid results in different properties of β-peptides, such that they exhibit 

higher stability against peptidases, and an enormous potential for secondary structure formation, 

as documented in numerous comprehensive reviews (e.g. [1]). Recently, we are studying different 

aspects of vibrational spectroscopy of small biological molecules of pharmaceutical interest such as 

halouracils [2], lactic acid [3], cystein [4], and β-alanin [5]. Analysis of literature data devoted to 

isoserine has shown that knowledge about physicochemistry and spectroscopy of this molecule 

important to biology and pharmacy is diminutive. This is why this is the first systematic study on 

conformers and IR spectroscopy of isoserine molecule. The IR low-temperature Ar matrix spectra 

of L-isoserine were registered for the first time and interpreted by means of the anharmonic DFT 

frequencies calculated at the B3LYL/aug-cc-pVTZ level. 54 L-isoserine conformers were calculated 

to be stable at the B3LYL/aug-cc-pVDZ and MP2/aug-cc-pVDZ levels. The most stable conformer 

and seven highly populated ones were recalculated by using the two methods and the aug-ccpVTZ 

basis set. The most stable conformer is dominating in the experimental spectra. A detailed 

interprpretation of the IR spectra of this conformer is performed based on comparison with its 

anharmonic B3LYL/aug-cc-pVDZ IR spectra and potenial energy distribution analysis. Inspection 

into the stretching vibration OH, C=O, and C-O regions of the experimental spectra enables one to 

detect at least four other L-isoserine conformers. The most stable conformer (1) is dominating 

whereas the other are much less populated is very visible in the ν(C=O) stretching vibration region 

of the spectra registered for Ar matrix. The presence of the conformer (2) in the Ar matrix IR 

spectra of L-isoserine can be proved based on the inspection into the β(OH) bending vibration 

region 1500-1200 cm -1. The side bands of the ν (C-O) band (1103 cm -1) of the conformer (1) 

positioned at 1121 and 1185 cm -1, corresponds the conformer (3). 

References 

[1] R. P. Cheng, S. H. Gellman, W. F. DeGrado, Chem. Rev., 101, 3219-3232 (2001). 

[2] J.Cz.Dobrowolski, J.E.Rode, R.Kołos, M.H.Jamróz, K.Bajdor, A.P.Mazurek, J. Phys. Chem.,109,2167-2182 (2005). 

[3] J. Sadlej, J. Cz. Dobrowolski, J. E. Rode, M. H. Jamróz, Phys. Chem. Chem. Phys., 8, 101-113 (2006). 

[4] J. Cz. Dobrowolski, M.ł H. Jamróz, R. Kołos, J. E. Rode, J. Sadlej, ChemPhysChem., 8, 1085-1094 (2007). 

[5] J. Cz. Dobrowolski, M. H. Jamróz, R. Kołos, J. E. Rode, J. Sadlej, ChemPhysChem., 9, 2042-2051 (2008). 

PA 44


Static and time resolved spectroscopy on 

bacteriophytochrome Agp2 

D. EHMER 1 , M. WOLF 1 , R. GROß 1 , J. LIN 1 , C. SCHUMANN 1+ , B. ZIENICKE 2 , T. LAMPARTER 2 AND R. 

DILLER 1 

1. Department of Physics, University of Kaiserslautern, Erwin- 

Schrödinger-Straße, Kaiserslautern, D-67663, Germany 

2. Botanical Institute I, University of Karlsruhe, Kaiserstraße 2, 

Karlsruhe, D-76131, Germany 

+ Current address: Leibnitz Institute for New Materials, Campus D2 2, 

Saarbrücken, D-66123, Germany 

Phytochromes are a wide spread family of photoreceptor proteins with two photointerconvertible 

forms, Pr and Pfr, absorbing in the red and far-red spectral region, respectively. First discovered in 

plants, meanwhile phytochromes have also been discovered in bacteria and fungi. The 

bathyphytochrome Agp2 from the plant pathogenic soil bacterium Agrobacterium tumefaciens 

behaves antagonistically to most other phytochromes. In darkness Agp2 converts from Pr into Pfr. 

The latter represents the thermal ground state, which is right in contrast to the second 

phytochrome Agp1 found in Agrobacterium [1, 2]. We present results of quasi-static UV-Vis 

spectroscopic measurements, which verify the thermal ground state being Pfr and a relatively fast 

dark reversion process unlike other bacteriophytochomes (Fig. 1a). For the first time we performed 

transient absorption experiments in the visible on the Pr form of Agp2 at microsecond time 

resolution to characterize the photocycle. Two fast time constants of about 0.1 and 0.7 ms (Fig. 1b) 

and another slower one were identified in analogy to Agp1 [3]. In addition we present first Vispump-Vis-probe-measurements 

on Pr with sub-picosecond time resolution. 

Fig. 1 – a) dark reversion process measured by UV-Vis-spectroscopy: in contrast to other phytochromes 

the Pr-maximum decreases and the Pfr-maximum increases, b) kinetics at 710 and 750 nm (Pr- and Pfrmaximum, 

respectively) at an observed time range of 2 ms and corresponding global analysis. 

References 

[1] B. Karniol, R. D. Vierstra, Proc. Natl. Acad. Sci. USA 100, 2807-2812 (2003). 

[2] K. Inomata, S. Noack, M. A. S. Hammam, H. Khwan, H. Kinoshita, Y. Murata, N. Michael, P. Scheerer, N. Krauss, 

T. Lamparter, J. Biol. Chem. 281, 28162-28173 (2006). 

[3] B. Borucki, D. v. Stetten, S. Seibeck, T. Lamparter, N. Michael, M. A. Mroginski, H. Otto, D. H. Murgida, M. P. 

Heyn, P. Hildebrandt, J. Biol. Chem. 280, 34358-34364 (2005). 

PA 45


Specific magnesium binding to RNA triplexes: 

Raman study 

SJ. ESPINOZA HERRERA 1 AND J. STEPANEK 1 

1. Institute of Physics. Charles University in Prague. Ke Karlovu 5, CZ- 

121 16 Praha 2, Czech Republic 

RNAs are negatively charged poly-ions, which need extramolecular positive charges in order to 

fold in different compact structures and to carry out their functions. Milimolar concentrations of 

divalent ions can stabilize RNA tertiary structures that are otherwise only marginally stable in the 

presence of high monovalent cation concentrations [1]. Magnesium, the divalent ion with the 

highest concentration in the intracellular space, is known to support selectively triple-helix 

formation in the mixed solution of poly(rU) and poly(rA) homopolynucleotides even if their 

stoichiometric ratio is optimal for the double-helix [2]. The mechanism of this phenomenon as well 

as details concerning the magnesium binding to RNA triplex was not before now resolved. In our 

study, we employed spectroscopic Raman titration to obtain spectral pattern caused by the specific 

magnesium interaction with the RNA triplex. Raman spectra of a set of poly(rU) + poly(rA) 

solutions with various magnesium concentrations were measured at conditions that guaranteed a 

full complexation of poly(rU) and poly(rA) chains into triplexes (2:1 poly(rU)/poly(rA) 

stoichiometric ratio; proper pH, ionic strength, and temperature). Raman spectra were treated by a 

factor analysis (singular value decomposition algorithm). The singular numbers show that the 

spectra in the series are composed basically by two spectral components. Dependence of the 

coefficient indicating mutual ratio of the two spectral components on the magnesium 

concentration corresponds to the McGhee-von Hippel model of a ligand binding to polymer; the 

size of the polymer binding site being equal to two U:A*U triads. The difference Raman spectrum 

displaying the effect of the specific magnesium binding reveals numerous peaks that (assigned 

according to known Raman markers [3]) do not correlate with the Raman spectral changes 

attributable to the triplex structure stabilization [4]. They reflect simultaneous direct binding of the 

magnesium surrounded by an incomplete shell of coordinated water molecules to adenine and 

uracil. Similar experiments performed for the poly(rC)/poly(rG) system, which is known to be less 

sensitive to the salt concentration [5], has revealed a different mode of magnesium binding. 

References 

[1] E.D. Draper, RNA 10, 335-343 (2004). 

[2] B.I. Kankia. Nucleic Acids Res. 31(17), 5101-5107 (2003). 

[3] J.M. Benevides, M. Tsuboi, J.K.H. Bamford, G.J. Thomas. Biophys. J. 72, 2748-2762 (1997). 

[4] J. Hanus, J. Stepanek, P.-Y. Turpin, J. Bok. J.Mol.Struct. 480-481, 437-442 (1999). 

[5] E. Plum, D.S. Pilch. Annu. Rev. Biomol. Struct. 24, 319-350 (1995). 

PA 46


Time-resolved photoacoustic spectroscopy of bacterial 

hemoglobins 

A. BOFFI 1 , A. FEIS 2 , C. GELLINI 2 , A. GUCCIONE 2 AND P.R. SALVI 2 

1. Department of Biochemical Sciences, University of Rome “La Sapienza”, Italy. 

2. Department of Chemistry, University of Florence, Italy. 

email: feis@chim.unifi.it 

We have applied time-resolved photoacoustic spectroscopy to the study of native and mutated 

“truncated” hemoglobins from the actinobacterium Thermobifida fusca. Photoacoustic 

measurements on the photodissociation of the CO complexes of heme proteins can yield kinetic 

information (in a 10 ns – 10 μs range ) which complements laser flash photolysis data [1]. In 

addition, the measurements allow to determine both volume and enthalpy changes in the 

reaction(s) triggered by CO photodissociation. Th. fusca hemoglobin is a prototypical bacterial 

(class 2) hemoglobin. It has been identified in a thermophilic actinobacterium [2] and 

overexpressed. The protein function has not yet been understood. Possible physiological ligands 

are O2, NO, HS -, which can bind to the heme Fe in the protein active site. The heme cavity 

structural properties are mainly due to the polarity and H-bonding capability of the (distal) amino 

acids: Tyr(B10), Tyr(CD1), Trp(G8). Single, double and triple Phe mutants of these key residues 

have recently become available. This set of mutated proteins offers the opportunity to assess the 

importance of each amino acid in the dynamics and in the energetics of the CO photodissociation 

reaction. Preliminary results point to an overwhelming role for Trp(G8). This is in agreement with 

a recent study on a related bacterial hemoglobin from Bacillus subtilis [3]. 

References 

[1] R.W. Larsen, J. Mikšovská, Coord. Chem. Rev. 254, 1101-1127 (2007). 

[2] A. Bonamore, A Ilari, L. Giangiacomo, A. Bellelli, V. Morea, A. Boffi, FEBS J. 272, 4189-4201 (2005). 

[3] A. Feis, A. Lapini, B. Catacchio, S. Brogioni, P. Foggi, E. Chiancone, A. Boffi, G. Smulevich Biochemistry 47, 902- 

910 (2008). 

PA 47


Delayed luminescence spectroscopy of erythrosin B in 

the presence of bacterial bioluminescence enzymes 

M. A. GERASIMOVA AND M. G. FOMINA 

Dept. of Experimental and Medical Physics, Siberian Federal University, 

79 Pr. Svobodny, Krasnoyarsk, 660041, Russia 

The phosphorescence of dyes in aqueous solution at room temperature is an uncommon 

phenomenon [1] because its triplet state is quenched effectively by the oxygen. Then, only delayed 

fluorescence of dye can be observed. The addition of biopolymers in solution can produce the 

intermolecular complex with dye. In this case it is possible to observe the phosphorescence of dye. 

Similar experiments were carried out in different fluid and solid biopolymer matrices [2, 3, 4]. In 

major cases such measurements were performed in anaerobic conditions therefore the goal was to 

measure the phosphorescence of dye in aerobic conditions. The paper presents the spectral and 

kinetic probing of triplet T1 and singlet S1 states of 50 μM erythrosin B upon addition of the 

enzymes of bacterial bioluminescent system using flash lamp with 5 μs pulse width. The mixture 

of enzymes contains bacterial luciferase from Photobacterium leiognathi and NADH:FMNoxidoreductase 

from Benecia harveyi. Only the delayed fluorescence of erythrosin B was observed 

in a pure aqueous solution, which spectrum coincides with the steady-state fluorescence. The 

presence of enzymes reveals the second peak of 695 nm in the long-wavelength region attributed 

to phosphorescence. The intensity of dye phosphorescence increases sharply (up to 4-fold) with the 

increase of enzymes concentration, whereas the intensity of delayed fluorescence increases slightly 

(up to 1.3-fold). The relation of quantum yields of dye delayed fluorescence to phosphorescence 

Φdf/Φp was found to decrease as enzymes concentration increases. For the dye to enzymes 

concentration ratios of 5:1 and 1:1 the values of Φdf/Φp are 0.42 and 0.14 respectively. The 

excitation wavelength of 260 nm attributed to the high excited states of dye was proved to pump 

T1 state more efficiently (6.7-fold) in comparison with the excitation wavelength of 500 nm 

according to S1 state. It should be noted that the peak and spectra shape of dye phosphorescence in 

the presence of enzymes do not change, while the delayed fluorescence spectra have the red shifts 

(up to 13 nm). The delayed and steady-state fluorescence spectra of dye in the presence and 

absence of the enzymes red shifted, while the delayed fluorescence tends to broaden spectra. 

Hence the energy gap between S1 and T1 states of erythrosin B decreased (from 3415 sm − 1 to 

3197 sm − 1) with increased enzymes concentration, which facilitates the intersystem crossing. The 

linear correlation of erythrosin B phosphorescence intensity and tryptophan delayed fluorescence 

intensity was demonstrated. The average phosphorescence lifetime of erythrosin B bound to the 

enzymes was determined with double-exponential fitting of the intensity decay. The lifetime of 

phosphorescence is 4-fold shorter (~ 0.1 ms) than the lifetime of delayed fluorescence. The delayed 

fluorescence lifetime of erythrosin B does not change upon addition of the enzymes (0.4−0.6 ms), 

but depends on the excitation wavelength [4]. 

References 

[1] R. Duchowicz, M. L. Ferrer, A. U. Acuna, Photochem. Photobiol. 68, 494-501 (1998). 

[2] M. A. Gerasimova, A. G. Sizykh, N. S. Kudryasheva, Vestnik KGU 1, 58-70 (2005). 

[3] P. B. Garland, C. H. Moore, Biochem. J. 183, 561-572 (1979). 

[4] L. C. Pravinata, Y. You, R. D. Ludescher, Biophys. J. 88, 3551-3561 (2005). 

PA 48


FTIR spectral signature for antitumoral drug effect on 

prostate cell lines 

RÉGIS GASPER 1 , DERENNE A, MIJATOVIC T 2 ., KISS R. 3 , GOORMAGHTIGH ERIK 1 

1. Structure et Fonction de Membrane Biologique (SFMB), Université 

Libre de Bruxelles, Brussels 1050, Belgium 

2. Unibioscreen SA, 1070 Brussels, Belgium 

3. Service de Toxicologie, Université Libre de Bruxelles, Brussels 1050, 

Belgium 

The number of anticancer agents that fail in the clinic far outweighs those considered to be 

effective, suggesting that the selection procedure for progression of molecules into the clinic 

requires improvement. New drugs are usually evaluated for their potential to kill cancer cell lines. 

Yet, there is a tremendous interest to obtain a fingerprint of their mechanism of action on the cells 

as identification of new mechanisms could lead to the development of new drug families. 

Presently, unless a new drug had a large cytostatic effect, it would be discarded from further 

testing. In turn, we are missing a large number of interesting compounds. Screening for "modes of 

action" presents a technical challenge that is beyond the capability of conventional methods used 

in cellular or molecular biology. We suggest in the present work that the infrared (IR) spectrum of 

cells exposed to anticancer drugs could offers a unique opportunity to get a fingerprint of all the 

molecules present in the cells and to identify, with a high sensitivity, the metabolic changes 

induced by potential anticancer drugs. Recently, it has been shown that cardiotonic steroids (CS) 

inhibit all proliferation and induce cell death in tumour cell lines. Our group evidenced that 

metabolic perturbation induced by such a drug on a prostate cancer cell line (PC-3) at very low 

concentration (~nM) can be monitored by infrared spectroscopy. To offer any relevance in drug 

screening, FT-IR spectroscopy should give a very characteristic fingerprint of the mode of action 

induced by one drug. To test this specificity, the effect of 4 molecules with a really close chemical 

structure will be tested in the presence of prostate cell line PC-3. The cells were incubated during 6, 

12, 24 and 36hrs in the presence of CS before analysis. We recorded about 20 spectra from at least 7 

independent cultures to achieve a good statistics sampling. All the spectra were recorded on a 

diamond ATR crystal and each sample (about 30000 cells) was rapidly evaporated under N2 flow 

to form a homogenous film on the crystal. We used multivariate statistical analyses to shed light 

on the metabolic changes induced by the different drugs. As all the molecules belong to the CS 

family, we did not expect large differences in their mode of action. Yet, we show here that FTIR 

spectroscopy is sensitive enough to identify at least 3 different metabolic perturbation signatures 

among the 4 molecules. 

PA 49


Room temperature phosphorescence spectroscopy of 

fluorone dyes bound to bacterial luciferase 

M. A. GERASIMOVA 


79 Prospekt Svobodny, Krasnoyarsk, 660041, Russia 

Phosphorescence is usually not observed in fluid solutions at room temperature. This is because 

there exist many deactivation processes that compete with emission, such as non-radiative decay 

and quenching processes, and result in vanishingly small phosphorescence quantum yields in 

room-temperature aqueous solutions. When dyes are located in highly protected environments 

within macromolecules the collisional de-excitations of triplet states are reduced, and the internal 

motions of molecules are restricted. In addition, N2 and sodium sulfite are often used as the 

oxygen scavengers. Phosphorescence has been observed for dyes in various biopolymers, such as 

amorphous sucrose [1], micelles [2], bovine serum albumin [3], and sol-gel silica [4]. In the paper, 

time-resolved and steady-state fluorescence spectroscopy was used to study the energy 

redistribution of the first excited triplet and singlet states of fluorone dyes (eosin Y, erythrosin B, 

rose Bengal) bound to bacterial luciferase from Photobacterium leiognathi. The spectra of room 

temperature phosphorescence and delayed fluorescence of 50 μM dyes were obtained at excitation 

wavelength of 260 nm (fig 1a). The efficiency of dye phosphorescence signal increases as the 

luciferase concentration increases and depends on excitation conditions. The relation of Φph/Φdf 

correlates with biopolymer concentration (fig. 1b) and tryptophan delayed fluorescence. The 

obtained trends become more significant for erythrosin B, and even more − for rose Bengal. The 

energy gap S1-T1 evaluated for halogenated derivatives of fluorescein decreases with increasing 

luciferase concentration. Hence, increased spin-orbit coupling facilitates the intersystem crossing. 

References 

Fig. 1 – (a) Delayed luminescence spectra of erythrosin B free in solution and bound 

to luciferase. (b) Correlation of the relative quantum yields of phosphorescence to 

delayed fluorescence for fluorone dyes with biopolymer concentration. 

[1] S. Shirke, Y. You, R. D. Ludescher, Biophys. Chem. 123, 122-133 (2006). 

[2] N. J. Turro, K.-C. Liu, M.-F. Chow, P. Lee, Photochem. Photobiol. 27, 523-529 (1978). 

[3] P. B. Garland, C. H. Moore, Biochem. J. 183, 561-572 (1979). 

[4] S. K. Lam, D. Lo, Chem. Phys. Lett. 281, 35-43 (1997). 

PA 50


MbCO↔matrix reciprocal effects in low hydration 

amorphous saccharide systems: a FTIR study 

S. GIUFFRIDA 1 , G. COTTONE 1 AND L. CORDONE 1 

Dipartimento di Scienze Fisiche ed Astronomiche, Università di 

Palermo, Via Archirafi 36, Palermo, I-90123, Italy 

Saccharides, and in particular, trehalose are well known for their high efficiency in protecting 

biostructures against adverse environmental conditions. Experiments and simulations [1] on 

carboxy-myoglobin (MbCO) have shown that the protein dynamics is highly inhibited in a lowwater 

trehalose host medium, the inhibition being markedly dependent on the amount of residual 

water. Furthermore, a mutual protein↔matrix structural and dynamic influence (coupling) has 

been recently observed in systems of very low hydration, which indicated that, at least in these 

systems, the protein-solvent master and slave relationship does not apply [2]. To better characterize 

these reciprocal effects, we performed an Infrared Spectroscopy (FTIR) study on the stretching 

band of the bound CO molecule (COB) and on the Water Association Band (WAB) in dry 

amorphous matrices of various sugars (trehalose, maltose, sucrose, lactose, raffinose), at different 

protein/sugar ratios. Such bands have already been successfully exploited for the simultaneous 

study of the thermal evolution (20-300K) of the embedded biostructure and of the matrix; indeed, 

the COB is one of most studied protein spectroscopical marker, and the WAB a very sensitive 

probe for the low-water matrix. As for the COB, in all the systems, the reduction of sugar content 

is reflected in the progressive increase of the A0 component, as expected, and the extent of such 

increase is peculiar of the each sugar. At variance, the WAB shows an unusual dependence on the 

protein/sugar ratio, which we ascribe to changes in the strength of the hydrogen-bond network, 

due to the different local environments experienced by the water molecules. This behaviour allows 

to classify protein-sugar systems on the basis of the mutual protein-sugar interactions (or different 

protein-solvent coupling). We suggest a strong coupling is needed for an efficient protection of the 

protein. 

References 

[1] L. Cordone, G. Cottone, S. Giuffrida, G. Palazzo, G.Venturoli, C. Viappiani, Biochim. Biophys. Acta-Prot. Proteom. 

1749, 252-281 (2005) and references therein. 

[2] S. Giuffrida, G. Cottone, L. Cordone, Biophys. J. 91, 968-980 (2006) 

PA 51


Effect of cosolvents on preferential conformations of 

blocked dipeptides 

M. GLUŠIČ AND J. GRDADOLNIK 

Laboratory of Biomolecular Structure, National Institute of Chemistry, 

Hajdrihova 19, 1001 Ljubljana, Slovenia 

The elucidation of the protein folding mechanism and related protein stability are unsolved 

problems of fundamental importance in molecular and particular in medicinal biochemistry. 

Protein misfolding and aggregation are the cause of some mayor diseases as the acquired 

spongiform encephalopathies, Alzheimer’s disease, Parkinson’s disease, cystic fibrosis, and many 

cancers. Protein stability is the result of a balance between intramolecular interactions of protein 

functional groups and their interactions with the solvent environment. Adding cosolvents into the 

protein solution can modify this balance. Because of that are cosolvents useful in investigating the 

physical origins of protein folding and the interactions that stabilize protein structure. Therefore, 

we studied the influence of three cosolvents on the structure of the dipeptides in the form of Ac-X- 

NHMe, where X represents an amino acid. We applied cosolvents, which have different effects on 

the protein structure and stability. Trimethylamine-N-oxide (TMANO) stabilizes the 

conformations of proteins, urea denatures proteins and trifluoroethanol (TFE) enhances helix 

formation in protein parts that have an inherent helix-forming tendency. A recent NMR [1] and 

vibrational studies [2] of dipeptides have shown that dipeptides possess conformational 

preferences for the dihedral angles φ and ψ. To study the influence of TMANO, urea and TFE on 

the dipeptide conformation we analysed amide III region in Raman and infrared spectra of pure 

dipeptides in water and dipeptides in water with added cosolvent. These measurements were 

compared with the measured 3JHNα NMR coupling constants. We found three different types of 

conformational populations (PII, αR and β) in all examined dipeptides. The occupation of these 

three conformational states depends on the type of side chain. The PII is the prevailing 

conformation in alanine and leucine dipeptide; however threonine, isoleucine and valine 

dipeptides adopt predominately the β conformation. The least populated conformation of 

dipeptides in aqueous solution is αR conformation. The only exception is glycine dipeptide. 

Surprisingly, added cosolvents do not change the distribution of preferential conformations in 

dipeptides. The expected interactions between the cosolvents and dipeptides were analyzed by 

difference infrared spectroscopy, analyses of proton chemical shifts and measuring of the 

halfwidths of proton signals. 

References 

[1] F. Avbelj, S. Golič Grdadolnik, J. Grdadolnik, R. L. Baldwin, Proc. Natl. Acad. Sci. USA 103, 1272-1277 (2006). 

[2] J. Grdadolnik, S. Golič Grdadolnik, F. Avbelj, J. Phys. Chem. B 112, 2712-2718 (2008). 

PA 52


YY1 is an intrinsically unstructured protein 

A. GÓRECKI, P. BONAREK, F. GOŁĘBIOWSKI, M. DZIEDZICKA-WASYLEWSKA 

Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian 

University, Gronostajowa 7 str., 30-387 Krakow, Poland 

Human transcription factor Yin Yang 1 (YY1) is ubiquitously expressed (and conserved among 

mammals) C2H2-type zinc finger protein, which regulates various promoters including viral and 

protooncogenic genes. Unusually, it can act either as repressor, activator or initiator of 

transcription that probably depends on specific DNA sequence or cellular context. The 

experiments performed so far do not allow to elucidate the mechanism of YY1 action related to 

particular function. It is generally accepted that understanding of protein function strongly 

depends on determination of protein structure; therefore the lack of structural information 

concerning YY1 is the main handicap. However, using various complementary computational 

approaches, we predict that YY1 do not posses the features typical of ordered protein and it 

belongs to the class of intrinsically unfolded proteins (IUPs). Both, the omnipotence of YY1 and 

lack of reports on structural studies confirm our supposition. Using different biochemical and 

biophysical approaches, we show that YY1 is indeed unstructured in solution. Spectroscopic 

studies, using among others circular dichroism, fluorescence and dynamic light scattering 

performed for E. coli expressed YY1 will be presented. 

PA 53


Distribution of conformations sampled by the central 

amino acid residue in GXG peptides inferred from amide 

I’ band profiles and NMR scalar coupling constants 

A. HAGARMAN 1 , T. MEASEY 1 , D. MATTHIEU 2 , H. SCHWALBE 2 , R. SCHWEITZER-STENNER 1 

1. Department of Chemistry, Drexel University, 3141 Chestnut St. Philadelphia PA 19104 

2. Institut für Organische Chemie und Chemische Biologie, Johann Wolfgang Goethe 

Universität, 60439 Frankfurt, Germany 

The conformational propensity of individual amino acids in the unfolded state of peptides and 

proteins is the subject of ongoing deliberation. Recent research has mostly focused on alanine, 

owing to its abundance in proteins and its relevance for the understanding of helix coil 

transitions. In the current study, we have analyzed the amide I’ band profiles of the IR, isotropic 

and anisotropic Raman, and VCD profiles of a series of GXG peptides, X representing a subset of 

the naturally occurring amino acids, in terms of a conformational model which explicitly considers 

an ensemble of possible conformations rather than representative structures. [1] The distribution 

function is expressed as a superposition of two-dimensional Gaussian functions associated with 

polyproline II (PPII), β-strand, helix and turn like conformations. We utilized these distributions to 

simulate the amide I’ band profiles as well as a set of J‐coupling constants obtained from twodimensional 

NMR experiments. [2] The results of our analysis reveal a PPII fraction of 0.79 for the 

central alanine residue in GAG, which strongly corroborates the notion that alanine has a very 

high PPII propensity. We performed a similar analysis for X=E, F, S, V, K, L and M. Our results 

indicate that S, E, M, K and L exhibit an intermediate PPII propensity (0.46-0.64), whereas F and V 

exhibit a less pronounced PPII propensity (


Identification of binding interactions between 

myeloperoxidase and its antibody using SERS 

D.R. HANSBERRY 1 , N.S. BABU 1 , C. PATEL 1 , AND E.S. PAPAZOGLOU 1 

1. Dept. of Biomedical Engineering, Drexel University, 3141 Chestnut 

Street, Philadelphia 19104, USA 

Surface Enhanced Raman Spectroscopy (SERS) is a widely used spectroscopic method that can 

significantly increase the sensitivity of Raman spectroscopy and has demonstrated significant 

benefit in the identification of biological molecules. We report the use of SERS to differentiate 

native myeloperoxidase (MPO) and a MPO polyclonal antibody (pAb) from the bound 

immunocomplex of MPO/pAb. The SERS signal was enabled by gold nanoparticles attached to 

MPO, pAb and their immunocomplex using an excitation wavelength of 785 nm. The SERS 

spectrum of MPO resulted in a signal with strong peaks in the 1356-1379cm -1 and 1548-1615cm -1 

regions, which is in agreement with previous literature on the Raman spectrum of MPO. 

Comparative SERS spectrum analysis of MPO, pAb, and their immunocomplex provides insight to 

the significant peak shifts and intensity variations brought on by the conformational changes due 

to the formation of their immunocomplex. Several key areas have been identified and indicate 

specific amino acids being shielded from undergoing resonance while new amino acids residues 

are made visible in the immunocomplex SERS spectrum and may be a result of conformational 

binding. Our work demonstrates the capability of SERS to identify binding events and 

differentiate an immunocomplex from unbound compounds with direct applications in 

biosensing. 

PA 55


Development of a protein infrared spectral databank for 

proteomics research 

P.I. HARIS & J.A. HERING 

Faculty of Health & Life Sciences, De Montfort University, Leicester, LE1 

9BH, United Kingdom, E-Mail: pharis@dmu.ac.uk 

The advent of Fourier transform infrared (FTIR) spectrometers has led to a surge in the application 

of infrared spectroscopy for biological studies especially proteins. FTIR spectroscopy can be used 

to analyse protein secondary structure in aqueous solution as well in the solid state. Due to the 

rapid speed of spectral recording and subsequent interpretation of the data, infrared spectroscopy 

is potentially a valuable tool for proteomics research [1-2]. The availability of protein structural 

data that can be readily accessed by the scientific community is an important factor in advancing 

scientific research and is also helpful for publicising a particular technique. Thus for example, the 

Protein Data Bank (PDB) has been a powerful publicity tool for X-ray crystallography and NMR 

spectroscopy. Currently, there are over 50,000 protein structures deposited in the PDB. Although 

the databank became functional in 1972, only 13 protein structures were deposited in 1976. In 

contrast, the number of structures deposited in 2008 is over 6,000. The growth of this databank 

has been remarkable. Unfortunately, there is no equivalent databank of protein infrared spectra 

containing as few as 100 proteins. Over the last few years we have been engaged in contacting 

various infrared spectroscopists to send us their infrared spectra for inclusion in our PISD (Protein 

Infrared Spectra Databank) but this has met with some success. However, we are continuing our 

work in this area and are engaged in producing a user friendly databank that can be easily 

accessed by scientists who can upload and/or download spectral data with relative ease. 

Furthermore, the databank will be hosted in a web-site that will provide an opportunity for 

carrying out analysis on the spectral data especially for quantification of protein secondary 

structure. The site will provide an opportunity to provide access to diverse algorithms for 

quantification of protein structure and will include, amongst others, a neural network based 

algorithm developed by us [2]. Results of our work on the development of the databank and the 

methods for quantification of protein structure from infrared spectral data will be presented. 

References 

[1]. J.A. Hering, P.R. Innocent, P.I. Haris, Proteomics 4, 2310–2319 (2004) 

[2]. J.A. Hering, P.R., Innocent, P.I. Haris, Proteomics, 2, 839–849 (2002) 

PA 56


Rapid identification of apoptotic T-cells using infrared 

microscopy 

G. HASTINGS 1 , R. WANG 1 , P. FUCHS 2 , Y. HSU 3 AND J. HILLIARD 2 

1. Dept. of Physics and Astronomy, Georgia State University, Atlanta 

GA 30303 USA 

2. Dept. of Biology, Georgia State University, Atlanta GA 30303 USA 

3. Dept. of Mathematics and Statistics, Georgia State University, Atlanta 

GA 30303 USA 

Many anti-leukemic therapies are aimed at inducing or restoring apoptosis in malignant B or T 

cells. With knowledge of apoptosis induced spectral biomarkers we will be able to use 

spectroscopic techniques to assess how malignant cells respond (via apoptosis) to some of these 

new therapies. With that goal in mind we have used FTIR transflection microscopy to study 

apoptosis induction in acute lymphoblastic leukemia T-cells (T-ALL cells). Apoptosis was induced 

in T-ALL cells in three ways: 1) Apoptosis was induced biochemically by incubating cells in the 

presence of the anti-Fas antibody. Fas is a member of the TNF receptor super-family, and its 

activation results in the activation of caspase-8, which results in a caspase cascade that leads to 

apoptosis. 2) Apoptosis was induced physically by irradiation of the cells with UVC light. 3) 

Apoptosis was induced by hyper-osmotic shock by incubating cells in 1 M sorbitol. Following 

apoptosis induction cells were incubated for an additional 3 hours at 37 oC. Induction of apoptosis 

in cells gave rise to significant IR spectral changes. In addition, the spectral changes observed for 

anti-FAs and sorbitol induced apoptosis were different from that induced by UVC irradiation. In 

particular, significant differences were found between spectra of normal and apoptotic cells in the 

1100-1000 cm -1 spectral region, with a band at 1086 cm -1 being significantly decreased for cells 

where apoptosis was induced using anti-Fas and sorbitol. We also used partial least squares 

multivariate regression methods and ROC curve analysis to establish with what specificity and 

sensitivity we could spectrally discriminate between apoptotic and non-apoptotic T-ALL cells. 

PA 57


Does the disulfide bridge have an effect on the 

conformational properties of the peptide hormone 

somatostatin-14? 

B. HERNÁNDEZ 1 , C. CARELLI 1 , Y. M. COÏC 2 AND M. GHOMI 1 

1. Groupe de Biophysique Moléculaire, UFR SMBH, Université Paris 13, 

74 rue Marcel Cachin, 93017 Bobigny cedex France 

2. Unité de Chimie des Biomolécules, URA 2128, Institut Pasteur, 28 rue 

du Docteur Roux, 75724 Paris cedex 15, France 

To emphasize the role played by the S-S bridge in the structural features of somatostatin-14 (SST- 

14) [1], we have applied our previously described experimental protocol to this peptide hormone 

[2]. Newly recorded CD and Raman spectra of this cyclic peptide and its open analogue obtained 

by Cys→Ser substitution, are presented. CD spectra of both peptides recorded in aqueous 

solutions in the 100-500 µM concentration range are strikingly similar. They reveal principally that 

random conformers constitute the major population in both peptides. Consequently the S-S bridge 

has no structuring effect at submilimolar concentrations. In methanol, the CD spectrum of 

somatostatin-14 keeps globally the same spectral shape as that observed in water, whereas its open 

analogue presents a major population of helical conformers. Raman spectra recorded as a function 

of peptide concentration (5-20 mM) and also in the presence of 150 mM NaCl, provide valuable 

conformational information. All Raman spectra present a mixture of random and β-hairpin 

structures for both cyclic and open peptides. More importantly, the presence or the absence of the 

disulfide bridge, do not seem to influence considerably different populations of secondary 

structures within this range of concentration. However, Raman spectra of SST-14 reveal a peptide 

concentration effect on the flexibility of the S-S linkage, and consequently on that of its cyclic part. 

In conclusion, although the disulfide linkage does not seem to markedly influence the SST-14 

conformational features in aqueous solutions, its presence seems to be necessary to assure the 

flexibility of the cyclic part of this peptide, and to maintain its closed structure in lower dielectric 

constant environments. 

References 

[1] M. Pawlikowski, G. Meleń-Mucha, Curr. Op. Pharmacol. 4, 608-613 (2004). 

[2] G.Guiffo Soh, B. Hernández, Y.M.Coïc, F.Z. Boukhalfa-Heniche, M. Ghomi, J. Phys. Chem. B 111, 12563-12572 

(2007). 

PA 58


Spectroscopic investigations of nonlinear optical 

material Benzaldehyde phenylhydrazone 

I. HUBERT JOE AND C. RAVIKUMAR 

1 Centre for Molecular and Biophysics Research, Department of Physics, 

Mar Ivanios College, Thiruvananthapuram – 695 015, Kerala, India 

Infrared and Raman spectra in conjunction with quantum chemical computations, lead to precise 

information about the structure of molecules. In the present investigation, the detailed spectral 

analysis of the molecule in the crystalline state is taken up to understand the correlation of the 

NLO activity, charge transfer interactions of the molecule supported by using the scaled quantum 

mechanical (SQM) force field technique based on Hartree Fock (HF) level with standard 6-31G* 

basis set. Single crystals of benzaldehyde phenylhydrazone (BPH) were prepared in acetone 

solution by slow evaporation technique. The IR spectrum of BPH was recorded using Perkin Elmer 

FTIR spectrometer in the region 400 -4000 cm -1 with the samples in KBr pellets .The resolution of IR 

spectrometer was 4 cm -1. The NIR-FT Raman spectrum was recorded using Bruker RFS 100 FT 

Raman spectrometer in the region 3500-100 cm -1. The quantum chemical computations of BPH has 

been performed using Gaussian '98 program package [1] at HF/6-31G* basis set. Normal 

coordinate analysis has been performed to obtain full description of the molecular motion 

pertaining to the normal modes using the MOLVIB program version 7.0 written by Sundius [2]. 

The optimized molecular structure is shown in Fig. 1. From the optimized geometry, the two 

phenyl rings are essentially planar with the dihedral angles C2-C3-C9-N14 (-179.318°) and N14-N15- 

C17-C18 (178.012°). The calculated first hyperpolarizability of BPH is 3.011x10 -30 e.s.u which is 16 

times that of urea. The normal mode 8a has been observed in IR at 1606 and in Raman at 1604 cm -1. 

The strong bands observed at 1584 in IR and 1589 cm -1 in Raman are assigned to 8b mode. The 

enhanced intensity clearly demonstrates the higher degree of the conjugation between two rings. 

The strong bands observed in IR at 1497 cm -1 have contributions of the 19a mode. The 19b mode is 

observed as strong band at 1454 cm -1 in the IR spectrum and the counterpart in Raman spectrum 

appear as a medium band at 1450 cm -1. The simultaneous occurrence of 8a and 19b provide 

evidences for the charge transfer interactions. 

References 

Fig. 1 – Optimized molecular structure of BPH. 

[1] M. J. Frisch, et. al GAUSSIAN 98, Revision A9, Gaussian, Inc., Pittsburgh, PA, (1998). 

[2] T. Sundius, J. Mol. Struct. 218, 321 (1990). 

PA 59


The photophysics and –chemistry in eumelanin 

building block 5,6-dihydroxyindole-2-carboxylic acid 

A. HUIJSER 1 , A. PEZZELLA 2 , M. D’ISCHIA 2 , L. PANZELLA 2 , A. NAPOLITANO 2 , V. SUNDSTRÖM 1 

1. Dept. of Chemical Physics, Lund University, Box 124, S 22100 Lund, 

Sweden 

2. Dept. of Organic Chemistry and Biochemistry, University of Naples 

Frederico II, Naples, Italy 

The UV-dissipative mechanisms in eumelanin building block 5,6-dihydroxyindole-2-carboxylic 

acid (DHICA) in aqueous environment at various degrees of protonation have been studied using 

ultrafast (sub-ps) time-resolved fluorescence spectroscopy. In addition, the effect of deuteration on 

the decay kinetics has been investigated. The excited fully protonated molecule is concluded to 

decay by sub-ps intramolecular proton transfer, via the existing H-bond, from the carboxylic acid 

group towards the indole nitrogen, followed by (non)radiative decay to the ground state. The 

mono-anion with the carboxylic group deprotonated dissipates absorbed energy either via a) 

(non)radiative decay to the ground state or b) ns intramolecular proton transfer from the 5hydroxyl 

group towards the carboxylic group, followed by (non)radiative decay to the ground 

state. Optical excitation of the third ground state geometry, the mono-anion with the 6-hydroxyl 

group deprotonated, leads to sub-ps intramolecular proton transfer from the 5-hydroxyl towards 

the deprotonated 6-hydroxyl group, as also deduced from quantum-chemical calculations before 

[1], followed by (non)radiative decay to the ground state. These findings show that excited state 

intramolecular proton transfer, in combination with radiative and nonradiative decay, are 

commonly occurring UV-dissipative mechanisms in DHICA. These mechanisms might be the basis 

of a built-in photoprotective function. This knowledge is of prime importance for future studies on 

larger sub-units of the eumelanin pigment. [2] 

References 

Fig. 1 – Chemical structure and labeling of C atoms of 5,6dihydroxyindole-2-carboxylic 

acid (DHICA). 

[1] S. Olsen, J. Riesz, I. Mahadevan, A. Coutts, J.P. Bothma, B.J. Powell, R.H. McKenzie, S.C. Smith, P. Meredith, 

Journal of the American Chemical Society 129, 6672-6673 (2007). 

[2] A. Huijser, A. Pezzella, M. d’Ischia, L. Panzella, A. Napolitano, V. Sundström, The photophysics and –chemistry of 

eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid, in preparation. 

PA 60


Infrared spectroscopic investigations elucidating the 

origin of the CO ligand at the active site of [NiFe]hydrogenase 

PHILIPP HUMMEL 1 , INGMAR BÜRSTEL 2 , OLVER LENZ², NATTAWADEE WISITRUANGSAKUL¹, 

BÄRBEL FRIEDRICH², PETER HILDEBRANDT 1 AND INGO ZEBGER¹ 

1. Max-Volmer-Laboratory for Biophysical Chemistry, TU Berlin, Straße 

des 17. Juni 135, 10623 Berlin, Germany 

2. Institute of Biology / Microbiology, HU Berlin, Chausseestr. 117, 

10115 Berlin, Germany 

Hydrogenases catalyze the cleavage and production of H2 in various microorganisms [1]. One 

important subclass of hydrogenase possesses an active site consisting of one iron and one nickel 

atom coordinated by four cystein residues. In the catalytic process, the oxidation state of the nickel 

is changed, while the iron remains in a low-spin Fe(II) state. The persistant Fe(II) state is 

maintained by ligation of three diatomic ligands to the iron, namely two cyanides and one carbon 

monoxide. Considering the toxicity of these ligands, the assembly of the CO and CN ligands is 

conducted via a sophisticated maturation machinery composed of at least six auxiliary proteins [2]. 

FTIR spectroscopy represents a powerful tool for the investigation of hydrogenases, as the spectral 

region of the vibrations of the CO and CN - ligands (about 2150 to 1850 cm -¹) is not overlapped by 

any protein or water absorption bands. Furthermore, substrate-labeling experiments allow the 

selective assignment of the two ligand types on the basis of characteristic isotope-shifts of the 

respective vibrations [3-4]. The CN - ligands have been shown to be derived from carbamoyl 

phosphate, while the CO ligand originates from a different source, which still remains unclear [3- 

5]. In order to reveal the origin of CO, ¹³C labeling experiments were performed using FTIR 

spectroscopy on the regulatory [NiFe]-hydrogenase of Ralstonia eutropha as a model system. It was 

shown that externally added CO is incorporated into the active site if provided in ample amounts. 

Ambient concentrations of CO, however, are not sufficient to be incorporated into the active site of 

hydrogenase. We, therefore, conclude that the CO ligand must be derived from the cellular 

metabolism. 

References 

[1] Cammack, Frey, and Robson (2001) Hydrogen As a Fuel: Learning from Nature, Taylor and Francis Ltd., London 

[2] Böck et al., Adv. Microb. Physiol. 51, 1-71 (2006). 

[3] Reissmann et al., Science 299, 1067-1070 (2003). 

[4] Lenz et al., FEBS Lett. 58, 3322-3326 (2007). 

[5] Forzi et al., FEBS Lett. 58, 3331-3337 (2007). 

PA 61


Abnormal shifts in Raman spectra of deuterated 

cytidine and 6-azacytidine 

S. GARASEVYCH, M. IAKHNENKO, O. SLOBODYANYUK AND I. VASKIVSKYI 

Department of Physics, Taras Shevchenko National University of Kyiv, 

64, Volodymyrs’ka St., 01033 Kyiv, Ukraine 

Raman study of cytidine, its structural analogue an anomalous nucleoside 6-azacytidine (6-azaC) 

and cytosine both in crystalline state and in water and DMSO solutions was performed. It was 

revealed that some peaks in spectra of 6-azaC dissolved in D2O are shifted to high frequencies in 

contrast to the ones in H2O solution. In the table below wavenumber positions ν and shifts ∆ν for 

the most two prominent lines in Raman spectra of cytidine, 6-azaC and cytosine are listed as 

example of normal and abnormal Raman shifts. Such abnormal Raman shifts apparently are 

caused by substitution of H with D atoms in solvent and/or nucleoside molecule. To discriminate 

effect of deuterated solvent and deuteration effect of the nucleoside molecule itself we have 

accomplished recrystallization of 6-azaC and cytidine from their H2O and D2O solutions. It occurs 

that abnormal Raman isotopic shift in 6-azaC crystals recrystallized from D2O solution takes place 

too. It proves noticeable deuteration of the nucleoside molecule itself. Similar results were 

obtained for recrystallized cytidine. An increasing of mode frequency under increasing of reduced 

mass due deuteration may be explained by advance strengthening of mode force constants. The 

last may be caused by substitution of intramolecular H-bonds with D-bonds. This assumption is 

confirmed in part by calculations of Raman spectra performed both for free molecules of studied 

compounds and their water solutions using Gaussian 03 package. Both normal and abnormal 

shifts close to the experimentally observed were obtained in the calculated spectra of 6-azaC and 

cytidine. Additional arguments were derived from experiments and calculations for normal and 

fully deuterated benzophenone that have no H/D-bonds and for normal and fully deuterated 2,2´dihydroxybenzophenone 

that contain two adjacent OH/OD groups. We believe this is the first 

observation of abnormal Raman frequency shifts of intramolecular modes under deuteration. It 

has essentially different origin than recently reported blue-shift due to improper H-bonding CH Y 

interactions [1]. This work was supported by the Fundamental Researches State Fund of the 

Ministry of Education and Science of Ukraine (grant № F25/137-2008). 

References 

Raman peak positions ν and shifts ∆ν 

in H2O and D2O solutions, cm-1 Normal shifts Abnormal shifts 

Substance ν(H2O) ν(D2O) ∆ν ν(H2O) ν(D2O) ∆ν 

6-azacytidine 

exp* 

cal** 

759,6 

774,5 

748,8 

761,8 

10,8 1288,9 

12,7 1292,4 

1302,6 

1299,9 

-13,7 

-7,5 

cytidine 

exp 

cal 

784 

786 

773,5 

773,6 

10,5 

12,4 

1244 

1266,9 

1247,6 

1270,3 

-3,6 

-3,4 

cytosine 

exp 

cal 

786,8 

785,1 

777,3 

772,3 

9,5 

12,8 

1290,2 

1296,8 

1291,3 

1303,2 

-1,1 

-6,4 

* – experimental, **– calculated 

[1] C. Dale Keefe, Elizabeth A. L. Gillis, L. MacDonald, J. Phys. Chem. A 113, 2544-2550 (2009). 

PA 62


DFT prediction of molecular geometry and vibrational 

characteristics of 1,3,4,6-tetrathiapentalene-2,5-dione 

and its radical cation 

S. JAISWAL, M. KUMAR, R. SINGH, G. SRIVASTAV, P. SINGH AND R. A. YADAV 

Laser and Spectroscopy Laboratory, Department of Physics, Banaras 

Hindu University, Varanasi-221005, India 

The molecular properties of 1,3,4,6-tetrathiapentalene-2,5-dione (TTP-DO), an important building 

block of organic conductors, and of its radical cation (TTP-DO +) have been studied by using DFT 

method at B3LYP/6-311++g** level [1]. Our result shows that significant changes are noticed in 

the geometrical parameters and vibrational characteristics of TTP-DO as the result of its 

radicalization. The TTP-DO and its radical cation have planar structure with D2h symmetry. The 

C=C bond loses its double bond character and magnitude of the C=C stretching frequency 

decreases significantly by 217 cm -1 with increased Raman activity as a result of radicalization [2]. 

The magnitudes of the four C-S bond lengths which are attached to the oxygen atom is found to 

increase by 0.027 Å where the magnitudes of the remaining C-S bond lengths is found to decrease 

by 0.036 Å due to the radicalization process. The C=O bond lengths are calculated decrease 

slightly by 0.018Å and the C=O stretching frequencies are found to increase by ~ 80 cm -1 in TTP- 

DO + as compared to its neutral molecule. In contrast to the C=O stretching frequencies, the 

magnitude of the C=O in-plane and out-of-plane bending frequencies for the two molecules due to 

the radicalization process are unaffected. 

References 

Fig.1 - Front View of TTP-DO 

[1] M. J. Frisch et al., Gaussian 03, Revision C.02, Gaussian, Inc.Wallingford CT (2004). 

[2] C. Adamo, R. Arnaud, G. Scalmani, H. Muller, F. Sahli and V. Barone; J. Phys. Chem. B 103, 6863-6869 (1999). 

PA 63


Structural fluctuation of proteins revealed by terahertz 

time-domain spectroscopy 

O. KAMBARA, K. TOMINAGA 

Molecular Photoscience Research Center, Kobe University, 1-1, 

Rokkodai-cho, Nada, Kobe, 657-8501, Japan 

To understand the expression mechanisms of protein function, our knowledge of the dynamical 

aspects of the protein molecules must be deepen. Such a structural fluctuation, which is connected 

directly with the protein function, is known to have a characteristic vibrational frequency in the 

low-frequency region below 100 cm -1 . Terahertz time-domain spectroscopy (THz-TDS) provides 

absorption coefficient and refractive index in the wavenumber region above 3 cm -1 from the 

changes of signal intensity and phase shift, respectively. We introduce a temperature correlation 

value of a product of these values as the novel physical quantity; reduced absorption cross section 

(RACS) [1]. In physicochemical, RACS is considered as a product of IR activity and density of 

states (DOS). If we assume that the IR activity is constant in the low-frequency region, the RACS is 

proportional to the DOS. We use a hen egg white lysozyme (HEWL) as a model protein. First of all, 

THz spectrum of HEWL without any preparation is measured at ambient temperature and then, 

RACS of the HEWL is obtained. Fig. 1 shows the frequency-dependent RACS of HEWL in the 

double logarithmic plot. The spectrum is approximately proportional to the power of the 

wavenumber in this spectral region. The exponent from 12 to 24 cm -1 is 1.8 (RACS ‐ ν 1.8), which is 

consistent with the DOS obtained by incoherent inelastic neutron scattering in the same spectral 

region (1.5-3.0 meV) [2]. This result directly suggests that the RACS for protein in the same 

condition is proportional to the vibrational density of states. Temperature and hydration effect on 

the low-frequency dynamics of proteins will be discussed from the RACS standpoint. 

References 

Fig. 1 – Double logarithmic plot of reduced absorption cross section 

of HEWL versus frequency. 

[1] K. Yamamoto, K. Tominaga, H. Sasakawa, A. Tamura, H. Murakami, H. Ohtake, and N. Sarukura, Biophys. J. 89, 

L22–L24 (2005). 

[2] S. G. Lushnikov, A. V. Svanidze, and I. L. Sashin, JETP lett. 82, 30-33 (2005). 

PA 64


Multiway chemometric approaches for 

spectrophotometric analysis of creatinine in serum 

samples from patients with renal failure 

M. KHANMOHAMMADI AND M. RAMIN 

Chemistry Department, Faculty of Science, IKIU, Qazvin, Iran 

Quantification of creatinine is an important standard measurement to assess the function of 

kidney. Moreover, abnormal creatinine concentration is correlated with some kidney diseases, for 

instance; acute nephritis, acute or chronic renal failure, renal tubule defect and urinary tract 

infection. Low levels of creatinine in blood may also indicate a decrease in muscle mass, caused by 

disease, such as muscular dystrophy, some types of severe liver disease, low protein diets, 

pregnancy or by aging [1]. Most of developed methods for quantitative determination of 

creatinine, which would be applied in clinical activities, are primarily based on reaction with 

alkaline picrate, called "Jaffe Method". The reaction takes place in an alkaline medium between 

creatinine and picric acid to form an orange- red compound, which can be measured 

spectrophotometrically at 500 nm. This procedure is simple and inexpensive but Jaffe reaction is 

not specific for creatinine. However, this reaction is affected by more than 50 endogenous 

compounds. One of the main issues for improving the analytical methods is to use chemometrics 

techniques. There are several useful techniques, introduced in analytical routes, providing more 

reliable results e.g. second-order calibrations. These calibration techniques have been 

demonstrated to be powerful methods for extracting qualitative and quantitative information from 

three-way data arrays such as multivariate spectroscopic-kinetic measurements with 

spectrophotometers. The main advantage of three-way multivariate calibration is that it allows 

concentration information of an individual component to be extracted in the presence of any 

number of non-calibrated constituents. Therefore, it is highly useful to overcome analytical 

problems involving a complex matrix. It is well known that mathematical methods like parallel 

factor analysis (PARAFAC) or multivariate curve resolution-alternating least squares (MCR-ALS), 

used for second order calibration are capable of resolving the underlying profiles and the relative 

concentrations of each component in the system. Consequently, only a small number of standards 

of the analyte are required for analysis. Second-order calibration and multivariate spectroscopickinetic 

measurements in the visible region were proposed to improve the Jaffe reaction for 

creatinine assay. Quantitative determination of creatinine by spectrophotometry, utilizing the 

PARAFAC and MCR-ALS techniques were compared. It was concluded that both methods can be 

used for creatinine determination in human serum, while the accuracy of the method, evaluated 

through the root mean square error of prediction (RMSEP), was 0.69 and 0.91 for PARAFAC and 

MCR-ALS respectively. 

References 

[1] R. Perrone, N. Madias and A. Levey, Clin. Chem., 38, 1933–1953 (1992). 

[2] J. Ghasemi, A. Niazi, Analytica Chimica Acta 533, 169–177 (2005). 

PA 65


From discrete multi-exponential model to lifetime distribution 

model and power law fluorescence decay function 

BORYS KIERDASZUK 

University of Warsaw, Institute of Experimental Physics, Department of 

Biophysics, 93 Zwirki i Wigury St., 02-089 Warsaw, Poland 

borys@biogeo.uw.edu.pl 

Experimental and theoretical studies of the fluorescence intensity decays in biomacromolecular 

systems showed that under constraints of typical experiment fluorescence lifetime distribution is 

given by gamma function [1], which led to a power-like decay function I(t)=[(2-q)/τ0][1-(1q)t/τ0] 

(1/(1-q)). The factor (2-q)/τo results from normalization, and the mean decay time is given 

by =τ0/(3-2q). Decays are described by mean value of lifetime distribution (τ0) and one new 

parameter of heterogeneity (q) related to the relative variance of fluctuations of γ=1/τ around the 

=1/τ0 value: q-1=/ 2. Taking into account decay with N decay channels, the total 

decay rate γ is expected to be a sum Σγi of a number N of partial rates γ i . In that case, q=2/N+1, 

i.e. N=2/(q-1). It is worth noticing that the normalization of power-like decay function leads to 

constraint on the q values (q2). 

Furthermore, requirement of existence of the mean value of decay time =τ0/(3-2q) implies that 

1 P(γγ) 

1.5 

1.0 

0.5 

N=1 

N → ∞, q → 1 

N=2 

N=4 q=1.5 

N=16 q=1.125 

0.0 

0 1 

γ / < γ > 

2 3 

PA 66 

→1, the gamma distribution becomes the Dirac 

delta function, and decay function converges from 

power-like form to the single-exponential form. 

When the heterogeneity parameter value increases 

stepwise from 1 to 3/2, deviation from singleexponential 

form increases, and it is more 

pronounced for the tail part of each decay. The 

power-like function well fits complex 

(heterogeneous) as well as simple monoexponential 

decays, and describes fluorescence 

decay kinetics by the parameter of heterogeneity – 

objectively reflecting physical heterogeneity of the 

system, and the mean lifetime value from 

distribution – characterizing the average rate of the excited-state decay. Numerous examples 

illustrate applications of a new model to rational analysis of complex fluorescence decays of 

biomacromolecules [1-4], e.g. protein-ligand complexes [2], which led to identification of 

tautomeric forms selectively bound by the enzyme [2, 3]. The latter is of great importance for the 

studies of the mechanism of protein (enzyme) action as well as for more rational drug design. 

[1] J. Wlodarczyk, B. Kierdaszuk, Biophys J. 85, 589-598 (2003). 

[2] J. Wlodarczyk, G. Stoychev-Galitonov, B. Kierdaszuk, Eur. Biophys. J. 33,377-385 (2004). 

[3] J. Wlodarczyk, B. Kierdaszuk, Biophys. Chem.123, 146-153 (2006). 

[4] B. Kierdaszuk, J. Wlodarczyk, Eur. Biophys. J. 36, 253-259 (2007).


Cobalt- siroheme containing enzyme 

A. V. KLADOVA 1 , O. YU. GAVEL 1 , J. J. CALVETE 2 , Z. GOUVEIA 3 , S. TODOROVIC 3 , I. MOURA 1 , J. 

J.G.MOURA 1 , S.A. BURSAKOV 1,4 

1. REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade 

de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 

2. Instituto de Investigaciones Biomédicas, 46010 C.S.I.C., Valencia, Spain 

3. ITQB, Universidade Nova de Lisboa Av. da República – EAN, 2780-157 Oeiras, Portugal 

4. Departamento de Protección Ambiental, Estación Experimental del Zaidin (EEZ-CSIC), 18008 

Granada, Spain 

anna@dq.fct.unl.pt 

A cobalt- porphyrin containing protein (CoPf) was isolated from the sulphate-reducing bacteria 

Desulfovibrio gigas. CoPf was sequenced and characterized by different spectroscopic techniques. 

The sequence confirms that this protein is a free form of the γ subunit of desulfoviridin of D. gigas. 

As isolated the protein is EPR silent, suggesting the presence of diamagnetic Co 3+. According to the 

results of mass spectrometry and modification by iodoacetamide (IA) and vinylpyridine (VP), the 

apo- CoPf contains two cysteines linked by disulfide bridge and one of them is only accessible to 

IA and VP, in presence denaturing agent (5 M ClGu). The monomeric violet coloured protein 

contains 1 Co/molecule of protein in a non covalently bound Co (III) porphyrin-like cofactor and 

exhibits UV-visible spectrum with peaks at 279 nm, 420 nm and 590 nm with shoulders at 300 nm, 

395 nm and 550 nm. The frequencies of the vibrational modes observed in the resonance Raman 

spectra of CoPf clearly indicate that the porphyrin, that houses the Co(III) ion, is a siroheme. 

PA 67


Interaction of porphyrin/oligonucleotide complex with 

liposomes studied by drop coating deposition Raman 

spectroscopy 

E. KOČIŠOVÁ, M. PROCHÁZKA AND J. ŠTĚPÁNEK 

Charles University, Faculty of Mathematics and Physics, Institute of 

Physics, Ke Karlovu 5, Prague 2, CZ-121 16, Czech Republic 

kocisova@karlov.mff.cuni.cz 

Oligonucleotides – short strands of nucleic acid - represent potential tools in the effort to 

specifically modulate protein expression inside living cells [1]. Since they are not able alone to 

penetrate the cellular membrane, some delivery mediator should to be used. Water soluble cationic 

porphyrins that can bind to oligonucleotides and compensate their negative charge seem to be 

appropriate candidates for this purpose [2]. We have studied interaction of the 

porphyrin/oligonucleotide complex - cationic copper 5,10,15,20-tetrakis (1-methyl-4-pyridyl) 

porphyrin (CuTMPyP) and 15 bases long oligothymidylate (dT15) - with liposomes prepared from 

asolectin representing a realistic model of the nature biomembrane, by using drop coating 

deposition Raman (DCDR). DCDR method [3] is based on deposition of a small droplet of the 

sample (units of µL) on a Teflon-coated stainless steel surface (SpectRIM slides, Tienta Sciences) 

with almost no intrinsic Raman signal. The hydrophobic surface enables drying of a sample by the 

“coffee ring effect” when the resulting flow of a liquid in the evaporating droplet carries dispersed 

material to its edge where it forms a ring. This method enables to obtain by means of Raman 

confocal microscope spectra of biomolecules in original concentrations down to 1 µM [4]. Dried 

droplet of our studied system on the SpectRIM slide revealed partial separation of the free 

CuTMPyP/dT15 complex and the CuTMPyP/dT15 complex interacting with liposome. This 

enabled obtaining Raman spectra of particular components. Factor analysis of Raman spectra 

showed various spectral changes, the most pronounced of which appeared in the CH stretching 

vibration region (2800-3000 cm -1). They correspond to reorientation of lipid chains in liposome as a 

consequence of their interaction with the CuTMPyP/dT15 complex. 

References 

[1] J. Goodchild, Curr Opin Mol Ther. 6, 120-128 (2004). 

[2] P. Praus, E. Kočišová, O. Seksek, F. Sureau, J. Štěpánek, P.-Y. Turpin, Current Organic Chemistry 11, 515 – 527 

(2007) and references therein. 

[3] D. Zhang, Y. Xie, M. F. Mrozek, C. Ortiz, V. J. Davisson, D. Ben-Amotz, Anal. Chem. 75, 5703-5709 (2003). 

[4] V. Kopecký Jr., V. Baumruk, Vib. Spectrosc. 42, 184-187 (2006). 

PA 68


Time-resolved and steady-state study of FMN 

fluorescence quenching by halides 

M. A. GERASIMOVA AND K. V. KOSHKOV 


79 Prospekt Svobodnyi, Krasnoyarsk, 660041, Russia 

Introduction of heavy atoms increases spin-orbit coupling and thus favors intersystem crossing in 

a molecule [1], leading to fluorescence quenching [2]. In the paper, influence of the heavy halide 

ions on the photophysical and spectral properties of flavin mononucleotide (FMN) being a crucial 

biochemical cofactor is treated. Both time-resolved and steady-state fluorescence methods used in 

the research are helpful in understanding complex photophysical behavior. The single-exponential 

intensity decay of 5⋅10 − 6 M FMN in buffer is fit using lifetime of 4.73 ns, which agree with the 

previous works (4.70 ns) [3]. The presence of halides results in two-decay-time model. Increasing 

the potassium halides concentration from 10 − 3 to 10 − 1 M caused an additional redistribution of two 

lifetimes and a sharp decrease of FMN average lifetime. Namely, at halide concentration of 0.3 M a 

longer lifetime of two-component mixture attributed to FMN in buffer decrease 5.6–, 4.4–, 1.2–fold 

for I − , Br − , Cl − respectively. The fractional contribution of this component also decreases abruptly to 

8% for I − , to 36% for Br − , and to 95% for Cl − . A shorter lifetime of FMN (~ 2 ns) decreases to 0.42 ns 

for iodide and 0.63 ns for bromide. In the case of 0.3 M KI intensity decay indicates the domination 

of this shorter lifetime. Upon addition of halides to FMN there was a decrease in intensity and no 

shifts in maximum or shape of steady-state emission spectra. The quenching data based on the 

average lifetime as well as steady-state measurements were represented as Stern-Volmer 

dependencies (Fig. 1). The Stern-Volmer plots deviate from linearity toward the y-axis for TCSPC 

data, which indicates that another type of quencher emerges at highest concentrations of halide. 

Lifetime data analysis reveals a 14–fold increase of FMN intersystem crossing rate constant in the 

presence of I − , a sevenfold increase in the case of Br − . Hence, upon addition of 0.3 M halides the 

fluorescence quantum yield of FMN decreased from 0.26 [3] to 0.03 for I − and to 0.05 for Br − . 

References 

PA 69 

Fluorescence yield 

16 

12 

8 

4 

0 

I − 

Br − 

F0 

F 

τ0 

τ 

F0 

F 

τ0 

τ 

0 0.08 0.16 0.24 

Halide concentration, M 

Fig. 1 – Stern-Volmer plots for the halides quenching of FMN. 

[1] S. K. Lower, M. A. El-Sayed, Chem. Rev. 66, 199-241 (1966). 

[2] K. N. Solov’ev, E. A. Borisevich, Uspekhi Fizicheskikh Nauk 175, 247-270 (2005). 

[3] P. H. Heelis, Chem. Soc. Rev. 11, 15-49 (1982).


Mechanistic studies of the Ras-superfamily by timeresolved 

FTIR spectroscopy 

C. KÖTTING AND K. GERWERT 

Ruhr-Universität Bochum, Lehrstuhl für Biophysik, ND04/352, D-44780 

Bochum, Germany, carsten.koetting@rub.de 

We report on trFTIR [1] investigations on the Ras protein, which is mutated in 30% of the human 

tumors. Ras plays a central role in cell signaling pathways, transducing growth signals from the 

plasma membrane to the nucleus. Ras acts as a switch, transmitting the signal in an active GTPbound 

form and turning the signal off in an inactive GDP-bound form. The switch off is 

accomplished by GTP hydrolysis, which is catalyzed by Ras and can be further accelerated by 

GTPase activating proteins (GAPs). Mutations which prevent hydrolysis cause severe diseases 

including cancer. A highly conserved “arginine-finger” of GAP is a key residue [2]. We investigate 

the reaction of the Ras GAP protein-protein complex within the FTIR spectrometer by means of a 

photolabile trigger. Detailed information on the reaction mechanism was revealed utilizing 

isotopic labels of the nucleotide, the GTPase and the GAP [3]: We assigned the absorption of the 

arginine-finger and showed, the finger does not point into the binding pocket in the ground state 

[4]. Once the arginine-finger points into the binding pocket, cleavage of GTP is fast and as an 

intermediate H2PO4 − hydrogen-bonded in an eclipsed conformation to the β-phosphate of GDP 

was found [5]. It is in a position to either reform GTP or be released from the protein in the rate 

limiting step of the GTPase reaction. While the catalysis of GTP hydrolysis by Ras is entirely due to 

enthalpic effects, which are caused by a charge shift towards the β-phosphate of GTP [6], the GAP 

increases the entropy of activation, probably by pushing ordered water molecules out of the 

binding pocket into the bulk [4]. Ras undergoes distinct post-translational lipid modifications that 

are required for appropriate targeting to membranes. We established a system to monitor lipid 

modified Ras at a single lipid bilayer with ATR-FTIR spectroscopy [7]. With this setup we are able 

to observe the absorption of individual functional groups of lipid anchored Ras and we can 

compare the reactions of Ras in solution with the reactions of Ras at the membrane at the atomic 

level. 

References 

1. C. Kötting, K. Gerwert, ChemPhysChem 6, 881-888 (2005). 

2. A. Wittinghofer, Biol. Chem. 379, 933-937 (1998). 

3. B. Warscheid, S. Brucker, A. Kallenbach, H.E. Meyer, K. Gerwert, C. Kötting, Vib. Spec. 48, 28-36 (2008). 

4. C. Kötting, A. Kallenbach, Y. Suveyzdis, A. Wittinghofer, K. Gerwert, PNAS 105, 6260-6265 (2008). 

5. C. Kötting, M. Blessenohl, Y. Suveyzdis, R. Goody, A. Wittinghofer, K. Gerwert, PNAS 103, 13911 (2006). 

6. C. Kötting, K. Gerwert, Chemical Physics 307, 227-232 (2004). 

7. J. Güldenhaupt, Y. Adigüzel, J. Kuhlmann, H. Waldmann, C. Kötting, K. Gerwert, FEBS J. 275 5910-5918 (2008). 

PA 70


Evaluation of bone site and sex as parameters for the 

Fourier transform infrared spectroscopic study of 

normal bone. 

N. KOURKOUMELIS 1 AND M. TZAPHLIDOU 1 

1. Dept. of Medical Physics, Medical School, University of Ioannina, 

45110, Ioannina, Greece 

Bone at the molecular level, is described as a composite material consisting essentially of calcium 

phosphate crystals, water and soft organic material, mostly collagen, which surrounds the mineral 

crystals by an exceptionally dense filling [1]. These constituents are commonly referred as mineral 

and matrix components and are distributed in diverse patterns among different bone types. The 

mineral phase in calcified tissues plays a significant role mostly because it strongly affects their 

strength and quality. Parameters such as mineral crystal perfection, composition and size, vary 

considerably in relation to bone age, type and disorders or diseases. Furthermore, the mechanical 

strength of bone depends mainly on the state of the cortical bone [2]. In the present work we have 

incorporated two additional parameters, bone site and sex, in order to study their effect to mineral 

content (ash weight) and composition of the cortical bone. Ten female and male Wistar rats, eight 

months of age and cortical sections from three different bone sites, femur, tibia and forearm (ulna) 

were analyzed by FT-IR spectroscopy via integration of the PO4 3- v4 (500-650 cm -1), CO3 2- v2 (855-890 

cm -1) and Amide I (1600-1700 cm -1) absorption bands in order to assess mineralization, maturity of 

mineral crystal and collagen, carbonate accumulation and environment, and acid phosphate 

content [3]. Our results showed that all inorganic phases consist of poorly crystalline B-type 

carbonated apatite while mineralization and carbonate content was virtually unaffected for all 

samples. The same conclusion was extracted for the maturity of collagen fibrils. Statistically 

significant differences however, were discovered for the non apatitic environments of both acid 

phosphate (P


Spectral components in coelenterate bioluminescence 

and in photoluminescence of Ca 2+-discharged 

photoproteins 

N.KUDRYASHEVA AND N. BELOGUROVA 

Institute of Biophysics SB RAS, Akademgorodok, Krasnoyarsk, 660036, Russia 

Ca 2+-regulated photoproteins are responsible for bioluminescence of marine coelenterates. The 

photoprotein is a stable enzyme-substrate complex consisting of: a single polypeptide chain (near 

20 kDa, containing three EF-hand Ca 2+-binding consensus sequences), substrate molecules – 

coelenterazine (imidazopyrazinone derivative) and oxygen. The addition of calcium ions to Ca 2+regulated 

photoproteins results in light emission. This peculiar feature of the photoproteins serves 

the basis for their analytical application, mainly in monitoring of intracellular biomedical processes 

in the presence of calcium ions [1-2]. The reaction generates a protein bound product, 

coelenteramide, in its excited state. The excited coelenteramide relaxes to its ground state 

producing blue light, with a spectrum maximum being in the range of 465 – 495 nm depending on 

the photoprotein type. Bioluminescent and photoluminescent spectra of products of the 

bioluminescent reactions (Ca 2+-discharged photoproteins) are complex, wide, and asymmetric. In 

our work, bioluminescence spectra of photoproteins from marine coelenterates – jellyfish Aequorea 

victoria and hydroid Obelia longissima, and photoluminescence spectra of their Ca 2+-discharged 

photoproteins were deconvolved into spectral components. Resolution of the spectra was 

performed in a combined way involving the methods of (1) function increment based on Gauss 

distribution, (2) secondary derivative, and (3) optimization of spectral components’ parameters 

and minimization of divergence. Four spectral components were found in original bioluminescent 

and photoluminescent (excitation and emission) spectra. Spectral maxima and contributions of the 

spectral components into the original spectra were determined. The spectral components were 

attributed to four forms of coelenteramide – one unionized and three ionized forms. The 

differences in bioluminescence spectra and photoluminescence spectra of Ca 2+-discharged 

photoproteins were discussed with protonic environment of coelenteramide taking into 

consideration. Contributions of spectral components into the photoluminescence spectra of Ca 2+discharged 

obelin were found to depend on calcium concentration, but bioluminescence spectra 

were not. Dependence of the photoluminescence spectra on excitation (for emission spectra) and 

registration (for excitation spectra) wavelength have been revealed. Photoluminescence spectra of 

Ca 2+-discharged photoprotein obelin have been shown to serve as respective colored marker to 

monitor intracellular processes with a resource for color variation. 

References 

[1] E.S. Vysotski, J. Lee, Acc. Chem. Res., 37, 405-415 (2004). 

[2] J.R. Blinks, W.G. Wier, P. Hess and F.G. Prendergast, Prog. Biophys. Mol. Biol., 40, 1-114 (1982) 

PA 72


Characterisation of fatty acid composition of human 

hair follicle dermal papilla cells using Fourier transform 

infrared microspectroscopy 

K. LAU 1 , B. R. WOOD 2 , D. NAUMANN 3 , P. LASCH 3 , A. HERMELINK 3 , R. PAUS 4 AND V. DECKERT 5,6 

1. Interface Department, Institute for Analytical Sciences, Bunsen-Kirchhoff Str 11, 44139 Dortmund, 

Germany 

2. Centre for Biospectroscopy, Monash University, 3800 Victoria, Australia 

3. Robert Koch Institute, Nordufer Str 20, 13353 Berlin, Germany 

4. Klinik für Dermatologie, Allergologie und Venerologie, Universitätklinikum Schleswig-Holstein, 23538 

Lübeck, Germany 

5. Institut für Physikalische Chemie, Friedrich-Schiller-Universitaet Jena, 07743 Jena, Germany 

6. Institut für Photonische Technologien, 07745 Jena, Germany 

The hair follicles in the skin present a rich source of stem cells important in regenerative medicine. 

In particular, the mesenchymal stem cells (MSC) located in the mesenchyme – dermal papilla(DP) 

and connective tissue sheath(CTS)- of the hair follicle, have been shown to be multipotent¹ ,² and 

participate in the dermal regeneration during cutaneous wound healing³. So far, there are no 

definitive markers for the human hair follicle MSC apart from their nestin expression, hindering 

the progress of isolating, characterising and exploiting them in regenerative medicine. In order to 

develop a label-free and non-destructive method to identify the human hair follicle MSC, Fourier 

Transform Infrared (FTIR) microspectroscopy was employed. FTIR spectra of the DP in a human 

skin cryosection were recorded in reflection mode using a Bruker IR Scope II instrument. Based on 

the literature on fatty acid (FA) in stem cells 4-6, we postulated the following features may be linked 

to 'stemness': low overall lipid contents in line with their quiescent character; high saturated FA to 

unsaturated FA ratio and low lipid chain disorder, both of which are linked to reduced membrane 

fluidity. A chemical map based on the integrated intensity of the 2880-2848 cm -1 region showed 

very low lipid in the DP compared to the surrounding cell layers indicating the FA in the DP cells 

are attributed not to lipid vacuoles but mostly to cellular membranes. An unsupervised 

hierarchical cluster analysis (UHCA) was performed on the 1 st derivative of the spectra over 3099- 

2840 cm -1. By correlating the chemical map to the UHCA false colour map, the DP clusters were 

distinguished. Their corresponding average spectra showed the stem cell-related features 

mentioned above, e.g. a cluster in the periphery of the DP with particularly high saturated FA: 

unsaturated FA ratio (I2864-2840/I3120-3020); and a cluster was found to contain νsym and νassym CH2 

peaks (2920 and 2850 cm -1) shifted to lower wavenumber values, suggesting a higher state of lipid 

chains order. The results therefore demonstrate that it is possible to identify stem cells in the hair 

follicle with FTIR once we further increase the spatial resolution. 

References 

[1] C. A. B. Jahoda, C. J. Whitehouse, A. J. Reynolds, N. Hole, Exp. Dermatol 12, 849-859 (2003). 

[2] G. Richardson, E. Arnott, C. Whitehouse et al, J. Invest. Dermatol. Symp. Proc. 10, 180-183 (2005). 

[3] C. A. B. Jahoda and A. J. Reynolds, The Lancet, 358, 1445-1448 (2001) 

[4] L. DiMascio, C. Voermans, M. Uqoezwa et al, J. Immuno 178, 3511-3120 (2007). 

[5] A. J. Bentley, T. Nakamura, A. Hammiche et al, Mol. Vis. 13, 237-242 (2007). 

[6] L. Teboul, D. Gaillard, L. Staccini et al, J. Biol. Chem, 270, 28183-28187 (1995) 

PA 73


In vivo elution properties of doxorubicin loaded beads 

revealed by microspectroscopies 

J. NAMUR 1, M. WASSEF 2, S. CITRON 3, A. LEWIS 4, JM. MILLOT 1, M. MANFAIT 1, A. LAURENT 5,6 

1. MEDyC UMR CNRS 6237, Reims, France, 

2. Pathology, Lariboisiere Hospital, Paris France 

3. Radiology, Piedmont Hospital, Atlanta, GA, United States 

4. Biocompatibles UK Ltd, Farnham, United Kingdom 

5. Neuroradiology, Lariboisiere Hospital, Paris, France. 

6. Matière et Systèmes Complexes UMR CNRS 7057, Paris, France. 

Drug eluting beads (DEB) are polymeric calibrated microspheres measuring 100µm to 300µm in 

diameter, and loaded with the anticancer molecule doxorubicin (DOXO) [1]. They are used in the 

treatment of liver cancers [2,3] with the idea that, compared to intravenous chemotherapy, they 

can increase the local dwell time and concentration of drug inside the tumor. While DOXO-DEB 

have demonstrated they can provide a sustained release of drug for at several months in vitro [4], 

their elution properties have never been assessed in vivo. We aimed to apply Fourier transform 

infrared microspectroscopy (FTIR-MS) and microspectrofluorimetry on tissue sections of DOXO- 

DEB treated livers to determine in situ 1) the amount of drug still retained inside the DEB and 2) 

the distribution and the concentration of the drug in the tissue around the beads. In a pig liver 

model without any tumor, we first evidenced that DOXO-DEB can release their drug load in vivo 

for a period of at least 3 months after the injection and that the drug may diffuse up to a distance 

of 600µm from the bead. In liver tumors explanted from 6 patients after DOXO-DEB injection, we 

further confirmed that DOXO-DEB can provide levels of drug above cytotoxic threshold (>1µM) 

[5], with a maximum delivery in the first hours after implantation. High concentrations of DOXO 

are associated with necrosis of the tissue further supporting the efficacy of the drug eluting system 

in the killing of tumor cells. Microspectroscopies are a very valuable tool to evaluate in vivo 

elution properties of drug eluting systems. 

Fig. 1 – DOXO concentration profiles in the 

tissue around DEB in liver tumors resected 

at different time points after DOXO-DEB 

treatment. Dot line : cytotoxic level [5]. 

References 

[1] A. L. Lewis, M. W. Gonzalez, A. W. Loyd et al. J. Vasc. Interv. Radiol. 17, 335-342 (2006) 

[2] M. Varela, M. I. Real, M. Burrel et al. J. Hepatol. 46 (3), 474-481 (2007) 

[3] K. Malagari, K. Chatzimichael, E. Alexopoulou. Cardiovasc. Intervent. Radiol. 31 (2), 269-280 (2008) 

[4] M. V. Gonzalez MV, Y. Tang, G. J. Phillips et al. J. Mater. Sci. Mater. Med. 19, 767-775 (2008) 

[5] J. J. Chuu, J. M. Liu, M. H. Tsou et al. J. Biomed. Sci.14 (2), 233-244 (2007) 

PA 74


The effect of alkali metal ions on the electronic 

structure of p-anisic acid 

M. KALINOWSKA1, R. ŚWISŁOCKA 1,2 , E. REGULSKA 1 AND W. LEWANDOWSKI 1,2 

1 Department of Chemistry, Białystok Technical University, 

Zamenhofa 29, 15-435 Białystok, Poland 

2 College of Computer Science and Business Administration in ŁomŜa, 

Poznańska 141B Street, 18-400 ŁomŜa, Poland 

In order to understand the nature of the interactions of biologically important ligands it is 

necessary to carry out the physico-chemical studies of these compounds with their biological 

targets (e.g., receptors in the cell or important cell components). Results of this study make it 

possible to predict some properties of a molecule, such as its reactivity, durability of complex 

compounds, and kinship to enzymes. In this paper the effect of alkali metal cations (Li, Na, K, Rb, 

Cs) on the electronic and molecular structure of p-methoxybenzoic acid (p-anisic acis) was studied. 

Methoxybenzoic acids are known to be important inhibitors in growth of bacteria and they 

influence the catalytic activity of many enzymes [1]. From this point of view it is very important to 

study the effect of substitutents and metals on the above mentioned biologically important ligands 

[2]. In this work the experimental IR (in solid state and solution), Raman, UV (in solid state and 

solution) and 1H, 13C NMR spectra of p-methoxybenzoic acid and its salts were registered, assigned 

and analyzed. Some of the obtained results were compared with published data for o- and m-anisic 

acid as well as o- and m-anisates. The structures of anisic acid and Li, Na and K p-anisates were 

optimised at the B3LYP/6-311++G** level. The IR, 1H and 13C NMR spectra and NPA, ChelpG and 

MK atomic charges were calculated. 

References 

[1] Qing-Xi Chen, Kang-Kang Song, Ling Qiu, Xiao-Dan Liu, Huang Huang, Hua-Yun Guo, Food Chem. 91, 269-274 

(2005). 

[2] W. Lewandowski, M. Kalinowska, H. Lewandowska, J. Inorg. Biochem. 99, 1407-1423 (2005). 

PA 75


A differentiation of the affinity of uranium(VI) to 

phosphate and carboxylic groups in native phosvitin 

studied by ATR FT-IR spectroscopy 

B. LI, J. RAFF, G BERNHARD AND H. FOERSTENDORF 

Institute of Radiochemistry, Forschungszentrum Dresden-Rossendorf, 

P.O Box 510119, D-01314 Dresden, Germany 

The toxicity of the uranium to the living organisms is because of its heavy metal characteristic. 

Proteins, the fundamental component of all living cells and the key to their metabolism, undergo 

conformational changes upon the heavy metal complexation, thus loss their proper cellular 

function. In this study, phosvitin, a highly water soluble 34 kDa protein containing roughly 35 

phosphate groups and 29 carboxylic residues, 1 is chosen as an ideal model system for the 

spectroscopic investigation of the interaction of U(VI) with proteins allowing the differentiation 

between the U(VI)-phosphate and U(VI)-carboxylic complexation. For this purpose, two different 

U(VI) concentrations (10 − 4 M and 10 − 5 M) are set up at pH 4 with various amounts of phosvitin to 

acquire complexes with different U(VI)/phosphate group ratios. The aqueous solutions were 

investigated by ATR FT-IR spectroscopy. For the very first time, soluble protein U(VI) complexes 

are achieved in aqueous solution providing spectral evidence for U(VI) complexation by the 

unequivocal identification of the νas(UO2 2+) mode. The spectra of the soluble complex show that at 

a low U(VI)/phosphate ratio (1:10.2) U(VI) preferentially binds to the phosphate groups. 

Interestingly, the νas(UO2 2+) mode is found at 905 cm − 1 which is bathochromic shifted about 60 cm −1 

compared to the free uranyl ion 2 reflecting a strong coordination to several phosphate groups. 

With increasing U(VI)/phosphate ratio, U(VI) complexation to carboxylic groups is observed by a 

hypsochromic shift of the νas(UO2 2+) mode and characteristic bands of the νs(COO − ) and νas(COO − ) 

modes. At a higher U(VI)/phosphate ratio (10:1), complexation between U(VI) and carboxylic 

groups becomes dominant. From the observed frequency of this mode (925 cm −1) a typical bidental 

complexation to U(VI) by carboxylic group can be assumed. 3 In order to reduce the impact of the 

carboxylic groups on the U(VI) binding, phosvitin is modified using EDC. After subsequent 

incubation with 10 − 3 M U(VI) in aqueous solution at pH 4, the obtained IR spectra of the 

precipitated U(VI)-protein complex confirm this assumption. 

References 

[1] Byrne, B. M., Van het Schip, A. D., Van de Klundert, J. A. M, Arnberg,´A. C., Gruber, M., Ab, G. Biochemistry 

(1984) 

[2] Müller, K.; Foerstendorf, H.; Tsushima, S.; Brendler, V.; Bernhard, G. J. Phys. Chem. A (2009) Accepted. 

[3] Kakihana, M., Nagumo, T., J. Phys. Chem. (1987) 

PA 76


Charge transfer processes in potentially antioxidant 

benzopyranes: a SERS study 

N.F.L. MACHADO 1 , C. RUANO 2 , J.L. CASTRO 2 , J.C. OTERO 2 , M.P.M. MARQUES 1 

1. Molecular Physical-Chemistry R & D Unit, University of Coimbra, 

Rua Larga, 3005-535, Coimbra, Portugal 

2. Dept. of Physical Chemistry, Faculty of Sciences, University of 

Málaga, E-29071, Málaga, Spain 

Benzopyrane-type heterocyclic compounds are natural products, widely distributed in plants, 

which are presently being studied as promising antioxidant agents. In view of their stability and 

low toxicity, this kind of compounds may constitute a safe and reliable option for use as additives 

in food and pharmaceutical industries, since a great number of the antioxidants currently 

employed are being rejected due to their toxicity [1]. Furthermore, benzopyrane-derivatives such 

as 1,4-benzopyrones (also known as chromones) display recognised pharmacological properties, 

from anti-inflammatory to antimicrobial, antiallergic, antispasmodic and antitumor activities [2]. 

Therefore, the study of the mechanisms through which these compounds act, both in vitro and in 

vivo, is of the utmost relevance for understanding their biological role, allowing a rational design 

of novel and more efficient antioxidants for use as anti-inflammatory and anticancer agents. Since 

there is a close relationship between activity and structure, it is essential to gather accurate 

conformational data on the systems under investigation. The present study aims at achieving this 

goal, using SERS (Surface Enhanced Raman Scattering) in silver colloids for the study of several 

benzopyrane derivates, in combination with quantum-mechanical calculations. The latter yields 

the lowest energy conformers, their predicted vibrational spectra, as well as the Raman enhanced 

bands when the molecule is involved in metal-adsorbate resonant charge transfer process. The 

widely employed hybrid method B3LYP, which includes a mixture of HF (Hartree-Fock) and DFT 

functionals was applied. The harmonic vibrational frequencies and the force field were calculated 

for the lowest energy conformer of each compound, while the force gradients in the excited CT 

state were obtained for the corresponding radical of each molecule, thus allowing the calculation 

of the corresponding CT-SERS intensities [3,4]. This approach allow us to detect charge transfer 

processes taking place between the molecule and the metal in a particular SERS by combining the 

experimental results with the theoretical data and analysing the bands undergoing SERS 

enhancement. 

References 

[1] G. Williams et al., J Food Chem. Toxicol. 37, 1027–1038 (1999). 

[2] A. Foroumadi et al., Bioorg. & Med. Chem. Lett. 17, 6764-6769 (2007). 

[3] A. ten Wolde et al., J. Phys Chem. 98, 9437-9445 (1994). 

[4] F. Avila et al., J. Phys. Chem. C, , 113, 105-108 (2009). 

PA 77


Ultrafast dynamics in heme 

and in heme-CO adduct of Tf-trHb 

A. MARCELLI 1 , I. JELOVICA BADOVINAC 2 , P. FOGGI 1,3 , C. GELLINI 4 , A. FEIS 4 , P. R. SALVI 4 , 

G. SMULEVICH 4 , AND A. BOFFI 5 

1. LENS, University of Florence, Via N. Carrara 1, Sesto F.no (Fi), 50019, 

Italy, marcelli@lens.unifi.it 

2. Dept. of Physics, University of Rijeka, Omladinska 14, Rijeka, Croatia 

3. Dept. of Chemistry, University of Perugia, Via Elce di Sotto 8, Perugia, 

06100, Italy, and INOA-CNR, Florence, Italy 

4. Dept. of Chemistry, University of Florence, Via della Lastruccia 3, 

Sesto F.no (Fi), 50019, Italy 

5. Dept. of Biochemical Sciences, Università “La Sapienza” , Piazzale 

Aldo Moro 5, Roma, 00185, Italy 

Ligand photodissociation with ultrashort laser pulses in hemoproteins-CO adducts is widely 

employed to unravel the elementary dynamical processes related to the functional properties of 

these proteins [1]. The characterization of the rebinding processes following CO photolysis allows 

us to obtain information about the active site organization. If the ligand remains near the metal, the 

geminate recombination takes place; instead, if the ligand easily diffuses away, the chance of 

ligand-metal encounters decreases. Here we present the results of a study on truncated 

hemoglobin from Thermobifida fusca (Tf-trHb) investigated with femtosecond time-resolved UVvisible 

absorption spectroscopy. A fast geminate recombination following CO photolysis has been 

observed, suggesting that the ligand is confined within the heme pocket. The distal single 

TrpG8Phe and triple TyrB10Phe, TyrCD1Phe, TrpG8Phe mutants have been also investigated in 

order to understand the role of specific amino acids in the distal pocket. The photon energy in 

excess to dissociation is deposited initially into the heme, triggering a cascade of dynamical 

processes. The nature of the short lived intermediates generated instantaneously after photolysis is 

a topic of current interest in hemoprotein dynamics [2]. The time-resolved behavior of the Tf-trHb 

is discussed on the basis of electronic/vibrational results on heme [3] and porphyrin macrocycle 

[4]. 

References 

[1] J.-L. Martin, M. H. Vos, Annu. Rev. Biophys. Biomol. Struct. 21, 199-222 (1992). 

[2] X. Ye, A. Demidov, F. Rosca, W. Wang, A. Kumar, D. Ionascu, L. Zhu, D. Barrick, D. Wharton, P. M. Champion, J. 

Phys. Chem. A 107, 8156-8165 (2003). 

[3] J. R. Challa, T. C. Gunaratne, M. C. Simpson, J. Phys. Chem. B 110, 19956-19965 (2006). 

[4] A. Marcelli, P. Foggi, L. Moroni, C. Gellini, P. R. Salvi, J. Phys. Chem. A 112, 1864-1872 (2008). 

PA 78


Synthesis and vibrational characterization of 

dimethylsilanediol 

M.P.G. RODRÍGUEZ ORTEGA 1 , M. MONTEJO 1 , F. MÁRQUEZ 1 , A. MARCHAL 2 AND J. J. LÓPEZ 

GONZÁLEZ 1 

1. Dept. of Physical and Analytical Chemistry, University of Jaén, 

Campus Las Lagunillas, Ed. B3, Jaén, E-23071, Spain 

2. Dept. of Inorganic and Organic Chemistry, University of Jaén, 

Campus Las Lagunillas, Ed. B3, Jaén, E-23071, Spain 

Proteases are one of the largest enzyme family encoded by the human genome, and the reaction 

that they catalyze, the proteolysis, is of key importance in many physiological and pathological 

processes. Thus, the development of inhibitors of this type of enzymes has become an important 

target of pharmaceutical research.[1] In this field, peptidomimetics containing the silanediol group 

(silicon bio-isosteres), that have proven activity as inhibitors of metallo-proteases (namely, ACE 

and Thermolysin) and aspartic-proteases (e.g. the HIV protease) are being a matter of study.[2,3] 

Since the inhibition process occurs via the establishment of hydrogen bonds between the inhibitor 

and the active site of the enzyme, the growing interest of this type of compounds could be justified 

by the remarkable properties of the silanodiol group as acceptor/donor of hydrogen bonds, the 

stability of the diolic form of silanediols contrasting the intrinsic instability towards the cetonic 

form of their C analogs (gem-diols) and the slightly longer Si-OH distances comparing with the C- 

OH distances, among others. Besides, the incidence of intramolecular hydrogen bonding in these 

silanediol derivatives seems to play a role in the stabilization of their molecular structures and, 

further, in their biological activities. For these reasons, we think that a deep knowledge of the 

chemistry of the silanodiol group in environments of increasing complexity would result of capital 

interest to: (i) understand the relations between the molecular structure and the biological activity 

of these compounds; (ii) achieve a better comprehension of the inhibition process; and (iii) lay the 

foundations for the development of a methodology of rational design of silanodiol-based 

proteases’ inhibitors. Nevertheless, despite the growing interest in these species, there is a lack of 

either theoretical or experimental works dealing with their structural characterization, maybe 

because of the size of the systems and the complexity of their conformational space. For these 

reasons, to accomplish their study, studying systematically the molecular structures and 

vibrational spectra (as a tool for structural characterization) of relatively simple alkylsilanediols of 

moderate size, such as dimethylsilanediol, diethylsilanediol, diphenylsilanediol, etc., will result 

useful. Following this methodology, the goal of the present work has been the synthesis of the 

simplest possible alkylsilanediol, namely dimethylsilanediol, and the study of its relations 

vibrational spectra/structure by means of the combined use of quantum chemical calculations and 

IR and Raman spectroscopies. 

References 

[1] J-T. Nguyen, Y. Hamada, T. Kimura, Y. Kiso, Arch. Pharm. Chem. Life Sci. 341, 523-535 (2008) and references 

therein. 

[2] S. McN. Sieburth, C-A. Chen, Eur. J. Org. Chem. 2, 311-322 (2006) and references therein. 

[3] L. Nielsen, T. Skrydstrup, J. Am. Chem. Soc. 130, 13145-13151 (2008). 

PA 79


Semiempirical model for the combined analysis of 

structural and spectral observables of heme proteins 

M. P. MARZOCCHI AND A. BELSHI 

Dept. of Chemistry, University of Firenze, Via della Lastruccia 3, Sesto 

Fiorentino (FI), I-50019, Italy 

A comprehensive analysis of information available from absorption and resonance Raman (RR) 

spectra, and X-ray crystallography of heme peroxidases and mutants involving residues close to 

the heme has been recently published [1]. The extensive data of the ferric state show that the heme 

iron sixth coordination site is vacant or bound weakly to water. The conformation of the heme 

chromophore which is affected by both the peripheral substituents and heme-protein interactions 

can play an important role on the enzyme functionality [2]. An empirical relationship between the 

conformation of vinyl group in position 2 and the RR νC=C stretching frequencies (cm -1) has been 

established [3]. In the present work we propose a semiempirical model involving spectroscopic 

(electronic absorption and RR spectra) and structural (Fe-Ligand distances) observables which are 

function, f(τ2), f(τ4), and f(τN), of the torsion angles of the peripheral substituents, 2- and 4-vinyl 

groups, and the proximal imidazole of the histidine residue. The model is applied to plant 

peroxidases (Fig. 1, points 1-9), metmyoglobins (10-13), and a hemoglobin (14). The Cartesian f(τ), 

2D polar f(φ), and 3D polar f(θ,φ) functions are characterized by δ, the phase angle, defining the 

orientation of the torsion axis about the Fe-N axis and by the maximal and minimal values of the 

observable, taken as parameters. For δ=0, f(τ) shows maxima at τ = ± π/4, ± 3π/4 radiants, f(φ) 

points at the same angles, and f(θ,φ) represents the real angular part of the Fe 3dπ orbitals in terms 

of spherical harmonics. The combined analysis of spectral and structural data allowed us to find: a) 

the absorption and RR frequency from the Fe-ligand distance and viceversa (Fig.1), b) the 

disposition of the chain of atoms involved in electron conjugation, and c) the interations between 

the heme and protein helices, the helix axis orientations being related to the torsion axis 

orientations of the observable as indicated by the phase angles, δ. 

Fig. 1 – Observed and calculated frequencies of RR ν 3 mode (left) and distances r(Fe-O) (right) 

References 

[1] G. Smulevich, A. Feis, B.D. Howes, Acc.Chem.Res. 38, 433-440 (2005) 

[2] B. D. Howes, C.B. Schiødt, K.G. Welinder, M.P. Marzocchi, J.-G. Ma, J. Zhang, J. Shelnutt, G. Smulevich, 

Biophys.J. 77, 478-492 (1999) 

[3] M. P. Marzocchi, G. Smulevich, J. Raman Spectrosc. 34, 725-736 (2003) 

PA 80


Raman spectra of hemoglobin: Three excitation wavelengths 

M.POLAKOVS 1 , N. MIRONOVA-ULMANE 1 , I. SILDOS 2 , AND M. PÄRS 2 

1. Institute of Solid State Physics, University of Latvia, Kengaraga 8 LV-1063, Riga, Latvia 

2. Institute of Physics, University of Tartu, Rija 142 Tartu, Estonia 

Resonance Raman (RR) spectroscopy is a particularly sensitive probe for studying the electronic 

and structural properties of metalloporphyrin complexes including haemoglobin (Hb) and 

myoglobin. The interpretation of the intense spectra obtained from metalloporphyrin complexes 

has been based on vibronically induced scattering from the B (Soret) or Q states from the 

porphyrin macrocycle. Resonance Raman scattering from Hb occurs only at its surrounding 

ligands group because only this part of the molecule absorbs in the visible and near ultraviolet 

region. Thus it is possible to investigate exclusively vibrations of the four heme groups of Hb 

without interference by scattering of the surrounding globin or other parts of the red blood cell 

(RBC) or erythrocyte. In this technique, laser excitation within an electronic absorption band 

produces selective enhancement of Raman bands associated with vibrations of the protein. In the 

case of heme proteins, vibrations of the porphyrin ring are enhanced due to resonance with the ̟- 

̟* transitions which dominate near ultraviolet absorption spectra. (the Soret band) and the visible 

(the α-β band). Different Raman scattering bands are brought out by excitation in the regions of the 

Soret absorption band or of the α-β absorption bands [1-3]. In this paper, we report the application 

of the Raman technique to study hemoglobin (Hb) in single erythrocyte using three different laser 

excitation lines: ultraviolet (441.6), visible (514.5 nm) and near-infrared (NIR; 830 nm). The Raman 

spectra of the hemoglobin in single erythrocytes were recorded on ”Nanofinder-S” using the 441.6 

nm excitation line and on a Renishaw Via instrument using 514.5 nm excitation radiation from a 

argon laser system and 830 nm from diode laser. The Raman excitation line used in 441.6 nm 

cause resonance enhancement with absorption band 415 nm (Soret band). The local coordinate of 

this band (pyrrole half ring stretching vibration) involves principally C–N stretching. We report 

the unusual enhancement of several bands in Raman scattering spectra of hemoglobin at excitation 

line 830 nm compared with the 441.6 and 514.5 nm wavelengths investigated. The origin of 

unusual enhancement of Raman scattering bands of hemoglobin will be discussed. 

References 

[1] B. R. Wood and D. McNaughton, Biopolymers 67, 259–262, 2002 

[2] B. R. Wood and D. McNaughton, J. Raman Spectrosc. 517–523, 2002 

[3] K.Ramser, K. Logg Goksör J. Enger, M. Käll, D. Hanstorp, Journal of Biomedical Optics 9(3), 593–600, 2004 

PA 81


Vibrational spectroscopic investigation of efavirenz 

using density-functional theory 

S. MISHRA 1 , D. CHTURVEDI 1 , A. SRIVASTAVA 1 , P. TANDON 1 AND A.P. AYALA 2 

1. Dept. of Physics, University of Lucknow, Lucknow 226 007, India 

2. Departamento de Física, Universidade Federal do Ceará, C. P. 6030, 

60.455-900 Fortaleza, CE, Brazil 

Efavirenz, (S)-6-chloro-4(cyclopropylethynyl)-1, 4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin- 

2-one, is a novel anti HIV agent belonging to the class of the non-nucleoside inhibitors of the HIV- 

1 virus reverse transcriptase [1]. Experimentally, infrared and Raman spectroscopy and 

theoretically DFT based quantum chemical calculations have been used to understand the 

structural and spectral characteristics of efavirenz. Based on these results, we have discussed the 

correlation between the vibrational modes and the crystalline structure of the most stable form of 

efavirenz. Electrostatic potential surfaces have been mapped over the electron density isosurfaces 

to obtain information about the size, shape, charge density distribution and chemical reactivity of 

the molecules. A complete analysis of the experimental infrared and Raman spectra has been 

reported on the basis of frequency of the vibrational bands and potential energy distribution over 

the internal coordinates from DFT/6-311G computations and characteristic bands have been 

identified. Plots of infrared and Raman spectra of the molecule based on DFT calculations show 

reasonable agreement with the experimental spectra. 

References 

Fig. 1 – Structure of molecule Efavirenz. 

[1] J. C. Adkins, S. Noble, Efavirenz Drugs 56, 1055–1064 (1998). 

PA 82


FTIR spectroscopic investigations of Co(II)-dextran 

complexes by using D2O isotopic exchange 

Z. MITIC 1 , M. CAKIC 2 AND G. S. NIKOLIC 2 

1. Dept. of Pharmacy, Faculty of Medicine, University of Niš, Bul. dr 

Zorana ðinñića 81, RS-18000 Niš, Serbia 

2. Dept. of Chemistry, Faculty of Technology, University of Niš, Bul. 

Osloboñenja 124, RS-16000 Leskovac, Serbia 

In the field of biocoordination chemistry a lot of investigations are based on the synthesis and 

characterizations of different metal complexes of ligands they present in biological systems, or 

synthetic ligands, which will serve like the model-molecules for complex biomolecular structures. 

Bio- or synthetic ligands are mainly natural chemical compounds of macromolecular type. In this 

group of products of the special importance are chemical compounds of polysaccharide dextran, 

pullulan and inulin with cations of the different d-biometals (Cu(II), Co(II), Zn(II) and Fe(III)). It is 

well known that raw microbiological exopolysaccharides dextran and pullulan, are glucose 

polymers with the large molar mass from a few millions g/mol, with own toxic and antigen 

characteristics so that they are not of pharmaceutical importance. For commercial reasons raw 

polysaccharides were depolymerized to the products with adequate molar masses, with the aim of 

getting fractions with narrow molar mass distribution. FTIR spectroscopic techniques (RT-FTIR, 

LNT-FTIR, D2O-FTIR, and ATR-FTIR) are applying in the structure analysis of polysaccharides 

and synthesized complexes, as well as for the confirmation of suggested types of complex 

structure and for the testing of homogeneities of samples [1−5]. Complexes of Co(II) ion with 

reduced low-molar dextran (RLMD) derivatives Mw=5000−6000 g/mol, were synthesized in the 

water solutions, at the boiling temperature and at different pH values (7−13.5). Complexes of 

bivalent Co ion with RLMD, their spectroscopic RT-FTIR and D2O-FTIR characterization, as well as 

the spectra-structure correlation, was investigated in this work. The samples of Co(II) ion 

complexes with RLMD were deuterated (D2O, Merck) for 2 hours, at room temperature, in 

vacuum. FTIR spectra as an average of 40 scans were recorded at room temperature (RT) on a 

BOMEM MB-100 FTIR spectrometer (Hartmann & Braun, Canada) equipped with a standard 

DTGS/KBr detector in the range 4000−400 cm − 1 with a resolution of 2 cm − 1 by the Win–Bomem 

Easy software. FTIR investigation of Co(II)-RLMD complexes by D2O isotopic exchange proved to 

be a very sensitive method for determining OH group coordination and is related to the hydrogen 

bond strength. Results of our investigations points to the dextran and their complexes with Co(II) 

ion are crystal hydrate molecules. Correlation of physicochemical, spectrophotometric and 

spectroscopic investigations of these complexes, coordination chemistry of Co(II) ion, structure of 

exopolysaccharide chain, are suggesting different model structures of the synthesized complexes. 

References 

[1] M. Cakić, Ž. Mitić, G.S. Nikolić, Lj. Ilić, G.M. Nikolić, Spectroscopy 22(2–3), 177–185 (2008). 

[2] R.G. Zhbankov, V.M. Andrianova, M.K. Marchewka, J. Mol. Struct. 436−437, 637−654 (1997). 

[3] Ž. Mitić, G.S. Nikolić, M. Cakić, P. Premović, Lj. Ilić, J. Mol. Struct. 924–926, 264–273 (2009). 

[4] Ž. Mitić, G.S. Nikolić, M. Cakić, et al., Russ. J. Phys. Chem. 81(9), 1433–1437 (2007). 

[5] Ž. Mitić, G.S. Nikolić, M. Cakić, et al., XII <strong>European</strong> <strong>Conference</strong> on the Spectroscopy of Biological Molecules 

(ECSBM'2007), Paris France, 319 (2007). 

PA 83


Adsorbate-induced aggregation of silver colloids and 

irreversible loss of SERS signal due to irradiation 

P. MOJZEŠ, J. PALACKÝ, AND B. LÁSKOVÁ 

Institute of Physics, Faculty of Mathematics and Physics, Charles 

University in Prague, Ke Karlovu 5, Prague 2, CZ-12116, Czech Republic 

Temporal evolution of the SERS spectra of a cationic porphyrin CuTMPyP4 was studied as a 

function of aggregation state of the borohydride-reduced Ag colloids. As the CuTMPyP4 is known 

to be an effective aggregating agent and excellent SERS-reporting molecule [1], using Ag colloids 

differing in surface potential and/or various concentration ratio between the colloidal particles 

and adsorbate molecules, different aggregation states can be finely tuned. Laser excitation at 441.6 

nm coincident with both the CuTMPyP4 Soret absorption band and the onset of plasmon 

absorption of nonaggregated as well as aggregated Ag colloids was selected to ensure comparable 

plasmon resonances for entire range of aggregation states [1]. SERS spectra were acquired as 

temporal series with short accumulation times (0.1 – 1 s), covering at a time spectral range 

including also stretching bands of bulk water (~3400 cm -1) employed as an internal intensity 

standard for precise SERS intensity normalization. It was found that normalized SERS intensity 

from macroscopic volumes of the colloids aggregated by addition of the CuTMPyP4 exhibits 

exponential decay in the course of continuous laser irradiation, similar to that reported recently for 

Au colloids [2]. Although SERS signal from more aggregated colloids was more intense 

immediately after exposure to the laser beam, it decreased rapidly to a constant level as the 

irradiation continued for dozens of seconds. No Raman signs of graphitic carbon or other 

photoproducts were identified in the spectra. Total intensity loss (up to 80% decrease) and decay 

rates (typically two-exponential; time constants τ1~10 s and τ2~100 s) were found to be related to 

aggregation induced by the absorbate. Conversely, SERS signal from colloidal systems exhibiting 

no apparent signs of aggregation on addition of the CuTMPyP4 remained constant even over longrun 

exposures. Signal decrease can be explained supposing two SERS contribution from the 

adsorbates localized at the sites of different morphology. The porphyrin fraction attached to the 

surface of isolated colloidal particles seems to be responsible for lower but exposure-time 

independent SERS signal. Molecules residing between aggregated colloidal particles, i.e. in the hot 

spots [2, 3], are exposed to considerably stronger electric fields [2, 3], their SERS enhancement is 

greater, however irreversibly disappearing with the exposure time. Similar loss of the signal was 

reported previously [3] at microscopic level as an abrupt termination of spectral and intensity 

fluctuations of single-molecule SERS due to irreversible photoinduced desorption of the adsorbate 

from the hot spot. Presence of two slightly different spectral forms of the CuTMPyP4 disclosed by 

multivariate statistical treatment of the time-evolving SERS spectra seems to indicate that spectral 

fluctuations in the hot spots or spectral differences due distinct microenvironments are not 

completely masked either in the macroscopic colloidal ensembles. 

References 

[1] M. Prochazka, P. Mojzes, B. Vlckova, P.-Y. Turpin, J. Phys. Chem. B 101, 3161-3167 (1997). 

[2] K. W. Kho, Z. X. Shen, Z. Lei, F. Watt, K. C. Soo, M. Olivo, Anal. Chem. 79, 8870-8882 (2007). 

[3] A. Weiss, G. Haran, J. Phys. Chem. B 105, 12348-12354 (2001). 

PA 84


Raman Spectroscopy for Arsenic Speciation 

D. GLORIA 1 , D.B. HIBBERT 1 , G. MORAN 1,2 , S. FOSTER 3 AND W. MAHER 3 

1. School of Chemistry, University of New South Wales, Sydney, NSW 

2052, Australia 

2. UNSW Analytical Centre, University of New South Wales, Sydney, 

NSW 2052, Australia 

3. Institute for Applied Ecology, University of Canberra, Canberra, ACT 

2601, Australia 

Arsenic speciation is of considerable importance in environmental toxicology, with effects ranging 

from very high toxicity for inorganic arsenite to low toxicity for arsenobetaine. In this work, we 

explore the potential of Surface Enhanced Raman Spectroscopy (SERS) as a technique for arsenic 

speciation. Few arsenic compounds have been characterized by Raman spectroscopy. Many of the 

important species of environmental significance are not commercially available and must be 

extracted and chromatographically purified from marine or other organisms. In collaboration with 

the Institute for Applied Ecology at the University of Canberra we are setting up a Raman and a 

SERS library of environmental arsenic compounds. We are also exploring SERS-based sensing as 

an approach to portable analysers for arsenic compounds. Nanostructured gold SERS substrates 

are prepared electrochemically and characterized by atomic force microscopy. Optimisation of 

SERS substrates is carried out using a two-level full factorial experimental design with aromatic 

thiols as test analytes. These gold surfaces are also being compared with commercial SERS 

substrates for application to arsenic analysis. 

PA 85


Unique perspective of FTIR-microscopy: Predicting 

relapse of colonic malignancy 

R. K. SAHU 1 , S. ARGOV 2 , S. WALFISCH 3 , E. BOGOMOLNY 1 , R. MOREH 1 AND 

S. MORDECHAI 1 * 

1. Department of Physics and the Cancer Research Center, BGU, Beer- 

Sheva, 84105, Israel 

2. Department of Pathology, SUMC, Beer-Sheva 84105, Israel. 

3. Colorectal Unit, SUMC, Beer-Sheva, 84105, Israel. 

This study aimed to determine the potential of IR-spectroscopy to diagnose abnormality in 

histologically normal resection margins for predicting relapse in colon cancer patients. The present 

study re-evaluates normal looking resection margins for abnormal crypt proliferation. Resection 

margins of 10 colon cancer patients (27 biopsies and 225 crypts in total), found completely normal 

by standard histology were reevaluated to identify signs of abnormality using Fourier transform 

infrared microscopy (FTIR-MSP). Absorbance in the region 900-1185 cm -1 arising from 

carbohydrates and nucleic acids was found to be the most effective variate for such evaluation. 

Discriminant Classification Function (DCF) analysis was also used in parallel to confirm the 

diagnosis. The abnormal crypts detected using the above optical method were correlated with a 

relapse in the patients’ history. The good agreement between the biopsy status as determined by 

the optical methodology and the relapse of colonic malignancy based on the patients’ medical files, 

establishes the potential of FTIR-MSP for medical purposes and hints at future clinical evaluation 

of the biopsies using this technique to predict relapse of colonic malignancy. 

References 

DCF value 

-15 

0 5 10 15 20 25 

PA 86 

15 

10 

5 

0 

-5 

-10 

Biopsy number 

Fig. 1 - Classification of biopsies based on the DCF scores to show 

abnormal and normal biopsies. Abnormal biopsies lie above the 0-0 

line and indicate a high risk of relapse for the patient. The sigmoid 

curve indicates a transition from normal to abnormal. 

[1] Sahu, R. K. et al. Detection of abnormal proliferation in histologically 'normal' colonic biopsies using FTIRmicrospectroscopy. 

Scand. J Gastroenterology, 39, 557-66 (2004). 

[2] Eastwood, G. L. Gastrointestinal epithelial renewal. Gastroenterology, 72, 962-75 (1977)


Gas-phase IR/UV spectroscopy of protonated antibiotic 

gramicidin S 

N. S. NAGORNOVA, T. R. RIZZO AND O. V. BOYARKIN 

Laboratoire de Chimie Physique Moléculaire, École Polytechnique 

Fédérale de Lausanne, Station-6, CH-1015 Lausanne, Switzerland 

Infrared (IR) spectroscopy of biomolecules in the gas-phase is capable to provide data for isolated, 

solvent free species, in the form of sets of vibrational frequencies. Each set, if measured on the 

same conformer of a molecule, constitutes a spectroscopic signature that can be used to test and to 

calibrate theory. We employ an IR-UV laser double resonance photo-fragmentation approach to 

measure conformer-selective IR spectra of protonated peptides, cooled to T=10K in a 22-pole linear 

ion trap [1, 2]. Deep cooling allows suppression of thermal congestion and drastically increases 

spectral resolution in UV and IR spectra. Here we study strongly bounded cyclic peptide 

gramicidin S. This molecule is a natural antibiotic, and its beta-sheet structures in solution and in 

crystal have been determined by NMR and by X-ray methods. In the gas phase it serves as a 

moderate size benchmark peptide to test calculations. We have measured IR-UV depletion spectra 

of gramicidin S in the finger print regions of amide I and amide II (C=O stretch, N-H bending 

vibrations) and N-H stretch [3]. We have also obtained similar spectra for a complex of this peptide 

with 18-crown-6 ether. This compound has a high proton affinity and it solvates charges on Orn 

side chains of gramicidin S, significantly shifting the near-by vibrational frequencies. Detection of 

these shifts facilitates assignment of the observed IR transitions. To finally sort out different 

vibrations we have recorded IR spectra in 15N isotopically labeled gramicidin-S and also in N-H 

deuterated species. We thus obtained a spectroscopic signature of gramicidin S that is to be 

reproduced by theory. Once validated the theory may predict the number of water molecules that 

make structure of a solvated gramicidin S similar to that measured in vivo. This prediction would 

challenge experiment to verify it through a measurement of IR spectra of the solvated species. 

References 

[1] O. V. Boyarkin, S. R. Mercier, A. Kamariotis, and T. R. Rizzo, J. Am. Chem. Soc. 128 (9), 2816 (2006). 

[2] J. A. Stearns, S. Mercier, C. Seaiby, M. Guidi, O. V. Boyarkin, and T. R. Rizzo, J. Am. Chem. Soc. 129 (38), 11814 

(2007). 

[3] N. S. Nagornova, T. R. Rizzo and O. V. Boyarkin, J. Am. Chem. Soc. 2009 (in preparation). 

PA 87


Spectral properties of the components of 

bioluminescent reaction in viscous media 

E. NEMTSEVA 1,2 , D. GULNOV 1 , E. ESIMBEKOVA 1,2 AND V. A. KRATASYUK 1,2 

1. Dept. of Physico-Chemical Biology, Siberian Federal University, 79 

Svobodny Prospect, Krasnoyarsk, 660041, Russia 

2. Lab. of Photobiology, Institute of Biophysics SB RAS, 50 

Akademgorodok, Krasnoyarsk, 660036, Russia 

Bioluminescent reactions are catalyzed by special enzymes called luciferases and emit light as one 

of the products. In luminous bacteria luciferase is coupled with NAD(P)H:FMN-oxidoreductase. 

The spectral properties of bacterial luciferase (Photobacterium leiognathi), oxidoreductase (Vibrio 

fischeri) and their substrate flavin mononucleotide (FMN) in viscous media were studied to reveal 

the mechanism of bioluminescence intensity regulation in cells. Different concentrations of 

glycerol, sucrose, gelatin and starch were used to modify the viscosity of micro- and macroenvironment 

of fluorophores. The following fluorescent characteristics of bacterial luciferase, 

oxidoreductase and FMN were measured: the emission and excitation spectra, the steady-state 

anisotropy and the lifetimes of excited states. Small shifts (up to 5 nm) of the protein emission 

spectra in the presence of glycerol, sucrose and gelatin were detected. Substantial change of 

fluorescence quantum yield for all components was registered. It was found that fluorescence 

lifetime of FMN is shorter in gelatin media and longer in glycerol and sucrose ones (in comparison 

with buffer solution). The data obtained were used to explain the change of bioluminescence 

intensity in modified viscous media: decreasing in the presence of glycerol and increasing in the 

presence of sucrose and gelatin. 

PA 88


Resistance to heavy metal ions- spectroscopic 

experimental approaches. 

C. NGONLONG E 1 , M. KHEIRO-DERFOUFI 1 , F. DE ANGELIS 1 , JM RUYSSCHAERT 1 AND G. 

VANDENBUSSCHE 1 

1. Dept. of Structure and function of biological membranes , Free 

University of Brussels, Boulevard du Triomphe, Brussels, 1050, Belgium 

The systems of transport play an important role in the ability of bacteria to grow in extreme 

biotopes such as soils highly contaminated by heavy metal ions. The proteins of the Superfamilly 

HME-RND (Heavy Metal Ion-Resistance Nodulation and cell Division) are notably involved in the 

transport of these ions out of cell [1].Cupriavidus metallidurans CH34 is a Gram negative βproteobacterium 

which is a model in resistance to heavy metal ions because it owns 12 putative 

HME-RND systems involved in the detoxification of Co, Zn, Cd, Cu, Ag. The Sil system has been 

recently identified in C. metallidurans CH34 by a two dimensional gel electrophoresis approach. It 

has been shown that the proteins are overexpressed by the strain in the presence of copper or 

silver in the culture medium. The inner and outer membrane proteins SilA (RND) and SilC (OMF) 

respectively and the periplasmic one SilB (MFP) form a tripartite complex. The RND protein is 

responsible for the substrate specificity and uses the dissipation of a proton gradient as driving 

force [2]. We have overexpressed and reconstituted into proteoliposomes, the RND protein SilA. 

Its structure and activity have been characterized by Infrared spectroscopy and fluorescence. We 

determined, by IR, the secondary structure of the protein and its orientation in reconstituted 

proteoliposomes by enzymatic digestion. The results are in accordance with previous structural 

data obtained for AcrB, the first HAE1-RND (Hydrophobic and Amphiphilic compounds) protein 

of the group that has been crystallized [3]. Furthermore, the fluorescence measurements showed 

that SilA can transport selectively copper ions in vitro when a proton gradient is imposed across 

the bilayer. 

References 

[1] Tseng T.T., Gratwick K.S., Kollman J., Park D., Nies D.H., Goffeau A., and Saier M.H. Jr. (1999) J. Mol. Microbiol. 

Biotechnol. 1, 107-125. 

[2] Elkins C.A., and Nikaido H. (2002) J. Bacteriol. 184,6490-6498. 

[3] Murakami S., Nakashima R., Yamashita E., and Yamaguchi A. (2002) Nature 419, 587-593. 

PA 89


The striking flexibility of the distal histidine in 

dehaloperoxidase 

F. P. NICOLETTI 1 , M. K. THOMPSON 2 , B. D. HOWES 1 , S. FRANZEN 2 AND G. SMULEVICH 1 

1. Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, 

I-50019 Sesto Fiorentino (FI), Italy 

2. Department of Chemistry, North Carolina State University, 2620 

Yarbrough Drive, Raleigh, North Carolina 27695, USA. 

Dehaloperoxidase (DHP) from Amphitrite ornata is a catalytically active hemoglobin-peroxidase, 

being able to oxidise phenolic substrates to quinone (1). Peroxidases are characterized by an 

increased polarity of the distal cavity compared to globins, however, a distal cavity Arg, which is a 

key residue in peroxidases promoting heterolysis of hydrogen peroxide during the catalytic cycle 

(3), is missing in DHP (2). It is well-known that heme pocket distal amino acid residues control 

ligand binding in hemoproteins. In fact, the comparison of the UV-Vis and resonance Raman (RR) 

spectra of the fluoride and hydroxide complexes of various peroxidases and selected mutants has 

highlighted the complex mechanism of stabilization of anionic ligands exerted by the distal amino 

acids. Moreover, this process resembles that of compound I formation during peroxidase catalysis, 

where ligand stabilization by the distal arginine is coupled to protonation of the distal histidine (4). 

It is of interest, therefore, to understand whether the different cavity characteristics of DHP, 

globins and peroxidases are also reflected in the binding of exogenous ligands. Since the 

spectroscopic study of the fluoride and hydroxyl adducts of heme proteins has furnished 

important information on how the distal heme protein cavity interacts with the exogenous ligand 

(4), in the present work we focus our interest on the RR and electronic absorption spectra of the 

fluoride and metaquo/hydroxyl adducts of DHP, and compare the results with those of 

myoglobin and peroxidases. In order to investigate the structural changes in the heme distal 

pocket upon binding of a substrate analog to DHP, the interaction of the ferric protein at pH 6.0 

with various mono-, di-, and tri-halogenated phenols has been studied. Comparison of the 

spectroscopic data with the available X-ray structures provides detailed insight into the role 

played by the distal histidine in the DHP reaction mechanism. 

References 

[1] J. Belyea, L. B. Gilvey, M. F. Davis, M. Godek, M. T. L. Sit, S.T. Lommel, S. Franzen, Biochemistry 44, 15637- 

15644 (2005). 

[2] V. de Serrano, Z. Chen, M.F. Davis, S. Franzen, Acta Crystallogr D Biol Crystallogr 63, 1094-1101 (2007). 

[3] A.T. Smith, N.C. Veitch Curr Opin Chem Biol 2, 2692-78 (1998). 

[4] G. Smulevich, A. Feis, B.D. Howes Acc Chem Res 38, 433-440 (2005). 

PA 90


Monitoring complexation of artificial peptide receptors 

by UV resonance Raman spectroscopy 

S. Niebling 1 , S. K. SRIVASTAVA 1 , P. C. WICH 2 , C. SCHMUCK 2 AND S. SCHLÜCKER 1 

1. Department of Physics, University of Osnabrück, Barbarastr. 7, 49076 

Osnabrück, Germany 

2. Department of Chemistry, University of Duisburg-Essen, 

Universitätsstr. 5, 45141 Essen, Germany 

Guanidiniocarbonyl pyrroles are artificial receptors for the efficient complexation of peptides even 

in polar solvents such as water [1]. Their carboxylate binding site (CBS) can be probed selectively 

by ultraviolet resonance Raman (UV RR) scattering. As this vibrational technique is extremely 

sensitive to small changes in bond lengths and force constants, it was chosen for a label-free 

binding study of these receptors with tetrapeptide substrates in water [2]. In solution there is an 

equilibrium between different species of the receptor, which is not only dependent on substrate 

concentration, but also on pH (Fig. 1). Studying the pH dependent UV RR spectra of the neat 

receptor was the first step of our Raman spectroscopic characterization. The corresponding acidbase 

equilibrium between the neutral and the protonated species is shown in Fig. 1. By using 

nonnegative matrix factorization (NMF), the corresponding component spectra could be 

determined. The experimental spectra are accurately modeled as a linear combination of the two 

component spectra [3]. The protonated receptor species is capable of effectively binding a 

carboxylate (e.g. the C-terminus of a peptide). By adding substrate to a receptor solution, the 

complexed receptor as a third species is formed (Fig. 1 right). A UV RR binding study at constant 

pH was performed in order to obtain the component spectrum of the complex. NMF component 

analysis yielded only two relevant spectra, one being the spectrum of the complexed species. The 

other component spectrum represents the sum of the unbound receptor species (protonated plus 

neutral form, constant ratio due to constant pH). Further studies will be focused on quantifying 

complexation of these receptors by tetrapeptide substrates. 

Fig. 1 – Acid-base (left) and complexation equilibrium (right) for guanidiniocarbonyl-based peptide 

receptors. 

References 

[1] C. Schmuck, Coord. Chem. Rev. 250, 3053–3067 (2006). 

[2] B. Küstner, C. Schmuck, P. Wich, C. Jehn, S. Srivastava, S. Schlücker, Phys. Chem. Chem. Phys. 9, 4598–4603 

(2007). 

[3] S. Srivastava, S. Niebling, B. Küstner, P. Wich, C. Schmuck, S. Schlücker, Phys. Chem. Chem. Phys. 10, 6770– 

6775 (2008). 

PA 91


SAXS and FTIR study on MbCO-Saccharide amorphous 

systems 

A. LONGO 1 , S. GIUFFRIDA 2 , M. PANZICA 2 , G. COTTONE 2 AND L. CORDONE 2 

1. Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), CNR, 

Via Ugo La Malfa 153, Palermo, I-90146, Italy 

2. Dipartimento di Scienze Fisiche ed Astronomiche, Università di 

Palermo, Via Archirafi 36, Palermo, I-90123, Italy 

Trehalose is a well-known bioprotecting disaccharide. Although its efficiency has been long 

assessed, the mechanism of trehalose-based bioprotection is still matter of debate. Many theories 

have been proposed, which rely on the role of water on the physical properties of the external 

matrix [1,2]. Here we report Small Angle X-Ray scattering (SAXS) and Infrared Spectroscopy 

(FTIR) results on carbonmonoxy-myoglobin (MbCO) embedded in amorphous matrices of 

trehalose and sucrose, at very low hydration level. SAXS data show the occurrence of ~15 nm local 

domains in protein-trehalose systems, which are absent in protein-sucrose systems and in the 

absence of protein. These domains become larger by increasing the sample hydration. The 

comparison between SAXS and FTIR data led us to assign this feature to protein-deprived regions, 

which better incorporate water than the protein rich background. We suggest that these domains 

might play a buffering role against the daily variations of atmospheric moisture in anhydrobiotic 

organisms, giving a hint for rationalizing the superior trehalose protective effects, in terms of 

structural properties of the whole protein-sugar system. 

References 

Fig. 1 – Pictorial view of samples from the SAXS results. A) 

trehalose/water: homogeneous domains are sketched in dark gray. 

B) sucrose/water: small size inhomogeneities are sketched in white. 

C) protein/trehalose/water: proteins are sketched in black, while 

trehalose/water domains are sketched in white. D) 

sucrose/protein/water:small size inhomogeneities are sketched in 

white; proteins are sketched in black. 

[1] J. H. Crowe, Adv. Exp. Med. Biol. 594, 143–158 and reference therein (2007) 

[2] L. Cordone, G. Cottone, S. Giuffrida, J. Phys.: Condens. Matter 19, 205110 (2007) 

PA 92


Spectroscopic study of marine microorganism adhesion: 

unraveling biomolecules and functional groups 

at the interface. 

L. PETRONE 1 , N. C. RAGG 2 AND A. J. MCQUILLAN 1 

1. Dept. of Chemistry, University of Otago, P. O. Box 56, Dunedin, New 

Zealand 

2. Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson, New Zealand 

All benthic marine organisms having a dispersal phase in their life histories require an adhesive 

strategy that allows them to form a strong and permanent bond to a range of substrates, under 

water, over a wide range of temperatures, salinities and conditions of turbulence. Algae and adult 

mussels are classical examples of permanent adhesion, secreting holdfasts to stick on virtually any 

hard surface in the tidal marine environment [1]. We present results for the adhesion study of the 

Perna canaliculus mussel larvae and Undaria pinnatifida algal spores. The presence of the 

bioadhesives was revealed by conventional scanning electron microscopy (SEM). Environmental 

scanning electron microscopy (ESEM) imaged the mussel larvae in its natural hydrated state, 

providing information on its hydrophilic nature. Energy dispersive X-ray (EDX) microanalysis was 

also performed, revealing in particular the presence of S, P and Ca in the mussel larvae adhesive 

[2]. Mg was detected in the algal spore adhesive. Additionally, algal spores and mussel larvae 

adhesion were studied by attenuated total reflection infrared (ATR-IR) spectroscopy. The infrared 

spectra showed protein and polysaccharide absorptions along with carboxylate, sulphated and 

phosphorylated vibrational modes. Spectroscopic studies on the adsorption of model compounds 

on a TiO2 surface confirmed the presence of sulphated, phosphorylated and carboxylated groups 

in the algal spore secretion. Such functional groups are responsible for the underwater initial 

interactions of the bioadhesives with the substratum surface, and the divalent cations are 

responsible of the cross-linkages of the bioadhesive leading to the formation of a gel-like adhesive 

[3]. Integrating the data obtained from surface chemistry, physical-chemistry and biochemistry 

experiments appear to be a necessary strategy to be able to elucidate the adhesion process of 

marine microorganisms in order to develop novel approaches to enhance the settlement, for 

instance, in the aquaculture industry or, on the other hand, to inhibit biofouling of underwater 

surfaces. 

References 

[1] K. J. Coyne, X. X. Qin, J. H. Waite, Science 227, 1830-1832 (1997). 

[2] L. Petrone, N. L. C. Ragg, E. Girvan and A. J. McQuillan, The Journal of Adhesion 85, 78-96 (2009). 

[3] L. Petrone, N. L. C. Ragg, A. J. McQuillan, Biofouling 24, 405-413 (2008). 

PA 93


Potential-dependend characterization of bombesin 

adsorbed states on roughened Ag, Au, and Cu electrode 

surfaces at physiological pH 

E. PODSTAWKA 1 AND G. NIAURA 2 

1. Regional Laboratory of Physicochemical Analysis and Structural 

Research, Faculty of Chemistry, Jagiellonian University, ul. Ingardena 3, 

30-060 Krakow, Poland 

2. Department of Bioelectrochemistry and Biospectroscopy, Institute of 

Biochemistry, Mokslininkų 12, LT-08662 Vilnius, Lithuania 

This work reports the direct surface-enhanced Raman spectroscopic (SERS) and Generalized Two- 

Dimensional Correlation Analysis (G2DCA) observations of the different orientations of the 

neurotransmitter bombesin (BN) chemisorbed on electrochemically roughened Ag, Au, and Cu 

electrode surfaces at different applied electrode potentials and at physiological pH [1]. The 

presence of the indole ring of Trp 8 and the amide bond between Gln 7 and Trp 8 of BN on these 

surfaces generates a specific SERS profile of BN adsorbed on the roughened Ag and Au electrodes 

that is affected by the electrode potential. Furthermore, for BN on Au, slight changes are observed 

in the band enhancement in comparison to what is observed for this neurotransmitter immobilized 

on Ag. In addition, there are larger changes in the spectra triggered by the substitution of Ag with 

Au electrodes and Ag with Cu electrodes than by substitution of Au with Cu electrodes. 

References 

Wavenumber / cm -1 

PA 94 

Raman intensity / cps 

160 

120 

80 

40 

0 

Ag 

575 

641 

641 

745 

758 

758 

836 

875 

875 

835 

882 

835 

757 

736 

760 

835 

878 

1004 

1002 

978 

1002 

1111 

1188 

1113 

1127 

1188 

1189 1125 

1010 

1114 

1303 

1259 

1298 

1258 

1304 

1272 

1034 

1114 

1127 

1235 

1277 

1012 

1663 

1607 

1565 

(a) 

(b) 

600 800 1000 1200 1400 1600 1800 

1356 

1355 

1344 

1342 

1439 

1439 

1530 

1563 

1606 

1658 

1439 

1530 

1557 

1604 

1432 

1666 

1668 

1593 

1550 

Fig. 1 - SERS spectra of BN adsorbed on a roughened Ag electrode 

at -1.200 V (a), -0.800 V (b), -0.400 V (c), and 0.000 V (d) 

potentials over the 500 – 1800 cm -1 spectral region. 

[1] E. Podstawka, G. Niaura, J. Phys. Chem. B submitted (2009). 

(c) 

(d)


Acesulfame-Potassium (additive E950) High-Intensity 

Non-Nutritive Sweetener: Computational and IR 

Studies on the Azanion, the Conjugated NH Acid and 

its Dimer 

A. D. POPOVA, E. A. VELCHEVA, I. G. BINEV 

Department of Structural Organic Analysis, Institute of Organic 

Chemistry with Center of Phytochemistry, Bulgarian Academy of 

Sciences, Acad. G. Bonchev Str., Bl.9, 1113 Sofia, Bulgaria 

6-Methyl-1,2,3-oxathiazine-4(3K)-on-2,2-dioxide (Acesulfame-potassium, Ace -K +) is a high 

intensity non-nutrive sweetener (Additive E950). The structures of Ace -, the corresponding 

conjugated NH acid Ace-H and its dimer (Ace-H)2 have been studied on the basis of both infrared 

spectra and density functional theory (DFT) computations. A good agreement has been found 

between theoretical and experimental spectroscopic characteristics of the species studied. 

According to both theory and experiment, like o-sulfobenzamide (saccharin), Ace-H exists as the 

dimer (Ace-H)2 in the solid state. The essential changes in the steric structure of Ace-H, caused by 

the conversion into Ace -, are spread over the whole sulfocarboximide group and the adjacent 

bonds. The new (azanionic) charge is distributed as follows: azanionic center (0.27 e -), carbonyl 

group (0.16 e -), –SO2O– (0.32 e -) and alkenylene group (0.25 e -). 

PA 95


Time-resolved microspectrofluorimetry and 

fluorescence imaging techniques in cellular uptake 

studies 

P.PRAUS 1 , E.KOČIŠOVÁ 1 , P.MOJZEŠ 1 , J. ŠTĚPÁNEK 1 , F.SUREAU 2 AND P.-Y. TURPIN 2 

1. Charles University in Prague, Faculty of Mathematics and Physics, Ke 

Karlovu 3, Prague 2, CZ-121 16, Czech Republic. 

2. ANBioPhy (Acides Nucleiques & Biophotonique) – UPMC/CNRS 

FRE 3207 GENOPOLE Campus 1, 5, rue Henri Desbruères 91030 EVRY 

Cedex, France 

Confocal microspectrofluorimeter has been adapted for time-resolved fluorescence measurements 

by using a phase-modulation principle and homodyne data acquisition method. This approach 

was employed to acquire intracellular spectra, which enabled us to determine lifetimes from 

selected intracellular sites. Modified oligonucleotides (ON) as sequences of chemically prepared 

deoxyribo- or ribonucleotides are able to regulate the gene expression [1]. Potential healing 

properties of ON are conditioned by effective cellular uptake. Synthetic cationic derivatives of 

porphyrins seem to be one of the promising candidates for this purpose. ZnTMPYP4 assisted 

delivery system is now studied to be used for ON intracellular transport. Time-resolved 

fluorescence spectra can reveal the ON and porphyrin interactions with present biomolecules [2]. 

Fluorescence confocal microimaging (Fig.1) has been complementarily employed to visualize 

penetration of the ON and porphyrin through cellular membrane and their distribution inside the 

cell. 

References 

Fig. 1 – Fluorescence image of 3T3 cell incubated with ATTO 425 

labeled dT 15 phosphorothioate complexed with cationic porphyrin 

used as delivery agent shows different intracellular distribution of 

ON (blue, artificial colors) and porphyrin (red). 

[1] J. Goodchild, Curr Opin Mol Ther, (2004), 6, 120-8. 

[2] P. Praus et.al., Ann. N.Y. Acad. Sci. 1130 (2008),117 – 121 

PA 96


SERRS microspectroscopy of porphyrins: improvement 

of sensitivity and spectral reproducibility 

M. PROCHÁZKA, P. ŠIMÁKOVÁ AND N. HAJDUKOVÁ-ŠMÍDOVÁ 

Charles University, Faculty of Mathematics and Physics, Institute of 

Physics, Ke Karlovu 5, Prague 2, CZ-121 16, Czech Republic 

prochaz@karlov.mff.cuni.cz 

Synthetic derivatives of porphyrins, natural forms of which are important biomolecules, are or can 

potentially be applied in photodynamic therapy of cancer, antiviral treatments, molecular biology, 

specific sensing of DNA sequences, selective cleavage of nucleic acids, etc. Porphyrins have been 

extensively studied by using surface-enhanced resonance Raman scattering (SERRS) spectroscopy, 

employing various metal surfaces including silver colloids [1]. This contribution is devoted to 

improvement of sensitivity and spectral reproducibility of SERRS microspectroscopy of 

porphyrins. We measured SERRS spectra of cationic free-base 5,10,15,20-tetrakis(1-methyl-4pyridyl) 

porphyrin (TMPyP) from gold or silver colloidal nanoparticles immobilized on glass (by 

silane [2,3] as well as by drying). Integrated confocal Raman microscopic system (LabRam HR800, 

Horiba Jobin-Yvon, with 632.8 nm and 514.5 nm laser lines) allowed us to improve sensitivity, to 

use short collection times and to scan Raman spectra point by point over the metal surface. SERS 

mapping provides very important information about spectral reproducibility of metal surfaces. We 

determined limits of detection (LOD) of TMPyP on gold and silver surfaces as ~ 5x10 -8 M and ~ 

1x10 -8 M, respectively. SERRS spectral mapping of gold immobilized nanoparticles shows their 

excellent reproducibility. In the case of silver immobilized nanoparticles, amorphous carbon with 

two broad bands at ~ 1300-1600 cm -1 and/or rapidly fluctuating peaks are frequently observed 

originating from decomposition of the adsorbate and/or silane or from contaminants adsorbed on 

the metal surface. Thus, these surfaces do not give reproducible data. In the case of silver 

nanoparticles, we therefore recommend to measure spectra directly from dried drops on pure 

glass. Surprisingly, dried drops of Ag colloid/TMPyP systems with varying TMPyP 

concentrations show the coffee ring effect similar to that used in drop coating deposition Raman 

(DCDR) technique [4]. This effect carries all nanoparticles to the edge where a ring from their 

aggregates is formed. If we measure from this ring, we obtain good SERRS spectra even in 1x10 -10 

M TMPyP concentration (by 1 s accumulation time) and also good reproducibility from point to 

point inside the ring. 

References 

[1] M. Procházka, J. Štěpánek, P.-Y. Turpin, J. Bok, J. Phys. Chem. B 106, 1543–1549 (2002) and references therein. 

[2] N. Hajduková, M. Procházka, J. Štěpánek, M. Špírková, Colloid Surf. A: Physicochem. Eng. Aspects 301, 264-270 

(2007). 

[3] N. Hajduková, M. Procházka, J. Štěpánek, P. Molnár, Vib. Spectrosc. 48, 142-147 (2008). 

[4] V. Kopecký Jr., V. Baumruk, Vib. Spectrosc. 42, 184-187 (2006). 

PA 97


Raman optical activity study of poly-L-proline chains of 

various lengths 

V. PROFANT 1 , V.BAUMRUK 1 AND P. BOUR 2 


University, Ke Karlovu 5, 121 16, Prague, Czech Republic 

2. Institute of Organic Chemistry and Biochemistry, Academy of 

Sciences, Flemingovo nám 2, 166 10, Prague, Czech Republic 

In this contribution we focus on the dynamic behavior of peptides mostly in aqueous solution 

where the peptide structural properties are most relevant to their biological role. In our experiment 

we measured Raman and Raman optical activity (ROA) spectra of oligo- and poly-L-proline 

samples in a wide frequency range and analyzed them with respect to the length of the proline 

chain. The relatively new technique of ROA [1,2] , which is based on different interaction of a 

specimen with right- and left-handed circularly polarized laser light, represents an ideal 

methodology for this type of observation. The stress is laid on the interconnection between 

experimental and theoretical approach, so the interpretation of observed phenomena is supported 

by ab initio calculations of spectral bands and their intensities. Our current study is strongly related 

to previous experiments [3] which were focused on the characterization of proline side chain 

conformation and its interaction with solvent. Measurements took place both in aqueous and in 

TFE solutions and we were able to obtain spectra of proline di-, tri-, tetra-, hexa-, nona- and 

dodekamer as well as of three commercial poly-L-proline samples of different mean molecular 

weight. 

References 

Fig. 1 – Poly-L-proline Raman (I R +I L ) and ROA (I R -I L ) experimental spectra measured 

in aqueous solution. The intensity scale expresses the total number of detected counts. 

[1] P.W. Atkins , L.D. Barron, Mol. Phys. 16, 453-466 (1969). 

[2] L.D. Barron, M.P. Boggard, A.D. Buckingham, Nature 241, 113-114 (1973). 

[3] J. Kapitan, V. Baumruk, P. Bour, JACS 128, 2438-2443 (2006). 

PA 98


Vibrational spectral analysis of biologically active 

crystal: Melaminium chloride hemihydrate 

C. RAVIKUMAR 1 , I. HUBERT JOE 1 AND V. K. RASTOGI 2 

Centre for Molecular and Biophysics Research, Department of Physics, 

Mar Ivanios College, Thiruvananthapuram – 695 015, Kerala, India 

2 Department of Physics, CCS University, Meerut, India. 

Melamine derivatives have attracted great attention in medical research, especially their antitumoral 

activity, used for the treatment of skin cancer. Also they are widely used in commercial 

field for making fibers and thermo setting plastics and industrial areas as a fire resistant foam [1,2]. 

Single crystals of Melaminium chloride hemihydrate (MCH) were grown by slow evaporation 

method and a vibrational spectral analysis was carried out using FT-IR and NIR FT Raman 

spectroscopy techniques. Ab initio quantum chemical computations were performed at the HF/6- 

31G(d) level to derive the equilibrium geometry, vibrational wavenumbers and intensities and first 

hyperpolarizability. Vibrational spectral analysis indicates the presence of peculiar N-H---O and 

N-H---Cl hydrogen bonding interactions. Hartree-Fock calculations reveal that protonation affect 

systematic changes in lengths of C-N bonds. N-H---Cl hydrogen bonding causes the shortening of 

donor-acceptor bond lengths and linearity of bond angles. The optimized molecular structure of 

MCH is shown in Fig. 1. The existence of intermolecular N-H---Cl hydrogen bonds due to the 

interaction between the lone pair of oxygen LP2Cl20 and LP2Cl37 with the antibonding orbital σ *N5– 

H19 and σ *N26-H36 has been confirmed by the results of NBO analysis. The splitting of Raman bands 

are observed at 107 and 104 cm -1 corresponds to N---O and N---Cl stretching H- bonds vibrations, 

through which the charge transfer can takes place inside the crystal providing the noncentrosymmetric 

crystal structure. 

References 

Fig. 1 – molecular structure of MCH. 

[1] Z. W. Wicks, P. N. Jones, S. P. Pappas, Film formation, compounds and appearance, Organic Coatings: Science 

and Technology, Vol. 1, Chap. 6, Wiley, New York, (1992). 

[2] A. J. Kirsch, N. Albrecht, C. Brogan, F. Lee, Amino coating resins: their invention and reinvention, Marketing 

communications Assoc. for CYTEC Industries, (1995). 

PA 99


The FT-IR, FT-Raman, 1H and 13C NMR study on 

molecular structure of sodium(I), calcium(II), 

lanthanum(III) and thorium(IV) cinnamates 

M. KALINOWSKA1 , W. LEWANDOWSKI 1,2 , R. ŚWISŁOCKA 1,2 AND E. REGULSKA 1 

1 Department of Chemistry, Białystok Technical University, 

Zamenhofa 29, 15-435 Białystok, Poland 

2 College of Computer Science and Business Administration in ŁomŜa, 

Poznańska 141B Street, 18-400 ŁomŜa, Poland 

The estimation of the electronic charge distribution in metal complexes allows more precise 

interpretation of the mechanism by which particular metals affect the chemical and biochemical 

properties of ligands as well as it makes possible to predict, what kind of deformation of the 

electronic system of ligands would undergo during complexation [1]. Cinnamic acid (3-phenyl-2propenoic 

acid), a derivative of phenylalanine, composes a relatively large family of organic acid 

isomers [2]. Cinnamic acid is extensively studied not only due to its important biological activity, 

but also because of its very specific structure. In the molecule of cinnamic acid the carboxylic 

group is being separated from the aromatic ring by a double bond. It causes the conjugations 

between the -C=C- bond and ̟-electron system. In this work the effect of sodium(I), calcium(II), 

lanthanum(III) and thorium(IV) ions on the electronic structure and molecular structure of 

cinnamic acid (phenylacrylic acid) was studied. In this research many miscellaneous analytical 

methods, which complement one another, were used: infrared (FT-IR), Raman (FT-Raman), 

nuclear magnetic resonance ( 1H, 13C NMR). The spectroscopic studies provide some knowledge on 

the distribution of the electronic charge in molecule, the delocalization energy of π-electrons and 

the reactivity of metal complexes. In the series of Na(I)→Ca(II)→La(III)→Th(IV) cinnamates: (1) 

systematic shifts of several bands in the experimental and theoretical IR and Raman spectra, and 

(2) regular chemical shifts of protons 1H and 13C nuclei were observed. 

References 


[2] L. Bravo, Nutr. Rev. 56, 317-333 (1998). 

PA 100


Polarised IR spectroscopy on macroscopically aligned 

amyloid fibrils. 

J. C. RODRIGUEZ-PEREZ , I. W. HAMLEY AND A. M. SQUIRES* 

School of Chemistry, University of Reading, Whiteknights, PO Box 217, 

Reading, RH6 6AH, UK 

E-mail: a.m.squires@reading.ac.uk 

Amyloid fibrils are self-assembled protein fibres related to some degenerative disorders like 

Alzheimer's or Huntington's diseases. The most used method for the study of protein structure, Xray 

crystallography, has had limited success with amyloids because of the difficulty to obtain 

crystals from them. In this report, we describe experiments where we align amyloid fibrils and 

study them with polarised vibrational (IR/Raman) spectroscopy. Such experiments can give new 

information about the structure and orientation of the peptide or protein chains within the fibril. 

We have used a number of methods to obtain aligned fibrils. These include “molecular combing”, 

a simple and inexpensive method where the receding meniscus of a liquid aligns the fibrils present 

in a solution in a direction parallel to its movement by means of solute-surface interactions. This 

technique has been used in other systems, for example, to obtain highly aligned strands of DNA. 

Here, we present results from three amyloid samples. These are made from the protein Lysozyme, 

and from two short synthetic peptides, TTR105-115 and YYKLVFFC [1]. In each case, the fibrils were 

successfully aligned, as revealed by the dichroic vibrational bands of their polarised IR spectra; 

and calculation of the orientational parameters of the groups involved in the vibrations was 

performed. We have also synthesised another peptide introducing a double isotope label (C 13, O 18) 

at two different positions to obtain residue specific information about the conformation of different 

parts of the peptide chain when it is assembled into amyloid fibrils. 

References 

[1] I. W. Hamley, V. Castelletto, C. M. Moulton, D. Myatt, G. Siligardi, C. L. P. Oliveira, J. Skov Pedersen, I. Abutbul, 

D. Danino, “Self-assembly of a modified amyloid peptide fragment: pH responsiveness and nematic phase 

formation”, Langmuir, submitted for publication (2009). 

PA 101


Molecular structure of calcium, magnesium, strontium 

and barium m-nitrobenzoates 

M. SAMSONOWICZ 

Department of Chemistry, Bialystok Technical University, 

Zamenhofa 29, 15-435 Bialystok, Poland 

Nitroaromatics are important priority pollutants entering the environment primarily through 

anthropogenic activities associated with the industrial production of dyes, explosives, pesticides, 

insecticides and pharmaceuticals. Discharge of these compounds into the environment poses 

serious health hazards as they are mutagenic and bioaccumulate in the food chain [1]. The changes 

in physical, chemical and biological properties of them decided about their effect on biological 

system. The aim of our works is the investigation of specific effect of various metals on the 

electronic system and molecular structure of some benzoic acid derivatives [2–5]. In this work the 

effect of calcium, magnesium, strontium and barium ions on the electronic structure of pnitrobenzoates 

was studied. The experimental IR (in solid state and solution), UV (in solution) 

spectra of m-nitrobenzoic acids and its salts were registered, assigned and analyzed. Characteristic 

shifts and changes in intensities of bands along the metal series were observed. The structures of 

m-nitrobenzoic and beryllium, calcium, magnesium, strontium and barium were optimised at the 

B3LYP/LANL2DZ level. Geometric aromaticity indices, NICS indices, atomic charges, dipole 

moments and energies were also calculated. 

References 

[1] I., Soojhawon, P.D. Lokhande, K.M. Kodam, K.R. Gawai, Enzeme Microb Technol 37, 527-533 (2005). 

[2] R. Śwsłocka, M. Samsonowicz, E. Regulska, W. Lewandowski, J. Mol. Struct. 6, 227-238 (2006). 


[4] R. Świsłocka, E. Regulska, M. Samsonowicz, T. Hrynaszkiewicz, W. Lewandowski, Spectrochim. Acta A 61, 2966- 

2973 (2005). 

[5] P. Koczoń, T. Hrynaszkiewicz, R. Świsłocka, M. Samsonowicz, W. Lewandowski, Vib. Spectrosc. 33, 215-222 

(2003). 

PA 102


Raman Spectroscopy, DFT calculations and thermal 

analysis as tools in host:guest interaction studies 

M. SARDO 1 , A.M. AMADO 2 AND P.J.A. RIBEIRO-CLARO 1 

1. CICECO, Chemistry Dept. University of Aveiro, Campus de Santiago, 

P-3810-193 Aveiro, Portugal 

2. Química-Física molecular, Chemistry Dept. University of Coimbra, 

P-3004-535 Coimbra, Portugal 

Different analytical techniques are commonly used to analyse the molecular structure and 

interactions established in a host-guest compound [1-3]. Moreover, the effects of the inclusion 

process on the guest molecules, in particular the conformational selectivity, have been the subject 

of several studies using Raman Spectroscopy [4,5]. In this work, studies were carried out into the 

effects of the inclusion process on α-, β- and γ-Cyclodextrins of a number of phenol derivatives (3- 

Methoxyphenol, 2-Methoxyphenol and 3-Methylphenol). The combined results from Raman 

spectroscopy, DFT calculations, DSC and TG analysis allow a consistent description of the 

inclusion compounds, providing further information on the inclusion process in CD hosts. The 

data collected from TG and DSC analysis suggest that the stoichiometry of the inclusion 

compounds ranges from 2:1 to 1:2, depending not only on the cavity size of the host but also on the 

structural features of the guest. For example, in the case of α-CD inclusion compounds, the 1:2 and 

1:1.5 stoichiometries were obtained for the meta substituted phenols while a 2:1 stoichiometry was 

observed for the ortho substituted derivative. The Raman spectra (Fig. 1) of the meta substituted 

guests clearly reveal the band splitting of several modes, consistent with the stoichiometry 

proposed in the thermal analysis section. This behaviour can be interpreted considering that there 

are two different chemical environments for the guest molecule: inside the CD cavity (A) and 

outside the cavity (B). 

References 

[1] S. Braga, et al., J Incl. Phenom. Macro. Chem. 43, 115-125 (2002). 

[2] J.C. Netto-Ferreira et al., Langmuir 16, 10392-10397 (1996). 

[3] T. Van Hees et al., Eur. J. Pharm. Sci. 15, 347-353 (2002). 

[4] P.J.A. Ribeiro-Claro et al., J. Raman Spectrosc. 27, 155-161 (1996). 

[5] A.M. Amado et al., J. Raman Spectrosc. 31, 971-978 (2000). 

PA 103 

A 

B 

B 

7 

0 

2 

6 

1 

2 

A 

5 

2 

B 

A 

239 249 

4 

2 

Wavenumber / cm-1 200 220 240 260 

αCD��3MePh 

3MePh


HPTLC-supported and in-situ resonance Raman 

spectroscopy of carotenoid signature in pollen 

F. SCHULTE1, 2 , J. MÄDER3 , L.-W. KROH3 , U. PANNE 1, 2 AND J. KNEIPP 1, 2 

1. Humboldt Universität zu Berlin, Chemistry Dept., Brook-Taylor-Str., 

D-12489 Berlin, Germany 

2. Federal Institute for Materials Research and Testing, I.42, Richard- 

Willstätter-Str. 11, D 12489 Berlin, Germany 

3. Berlin University of Technology, Institute of Food Technology and 

Food Chemistry, Department of Food Analysis, Gustav-Meyer-Allee 25, 

D-13355 Berlin, Germany 

The examination of carotenoids contained in tree pollen was accomplished by High Performance 

Thin Layer Chromatography (HPTLC) aided resonance Raman spectroscopy. Raman spectra were 

excited with 488 nm so that the laser light coincided with an electronic transition of the carotenoids 

and according signals were enhanced. Combination tones and overtones could be observed in the 

range from 2000 cm -1 to 4000 cm -1. The carotenoid signature of whole pollen grains was recorded 

and compared to Raman spectra of separated carotenoids. Extracts containing carotenoids derived 

from pollen were separated by HPTLC. For the separation of these carotenoids a new highly 

effective multiple development protocol for HPTLC was established using a gradient of 

methylenchloride, tetrahydrofurane and n-hexane as mobile phase. The information in the in-situ 

carotenoid signatures is confirmed to a great extent by the resonance Raman spectra of the 

HPTLC-separated carotenoids. In particular, shape and position of the ν1 signal around 1530 cm -1 

vary for different plant species and for particular carotenoids molecules. As the Raman scattering 

was excited in resonance (with an excitation wavelength of 488 nm), carotenoids were detected 

more sensitively with in-situ Raman spectroscopy than with HPTLC. Raman spectra were 

measured directly on the HPTLC plates with as less loss of time as possible to minimize the 

rearrangement of carotenoids as they are very sensitive to light induced isomerisation. The 

carotenoid composition of six tree pollen species was analysed (horse chestnut, large-leaved 

linden, european ash, sallow, mahaleb cherry, tree of heaven) and the concentrations of four 

prominent carotenoids (beta-carotene, cryptoxanthin, lutein, zeaxanthin) in these pollen species 

were determined. 

PA 104


Analysis of human skin cancer biopsies by IR 

spectroscopy and fuzzy clustering 

D. SEBISKVERADZE 1 , C. GOBINET 1 , E. LY 1 , M. MANFAIT 1 , P. JEANNESSON 1 , O. PIOT 1 AND V. 

VRABIE 2 

1. MéDIAN, CNRS UMR 6237 MEDyC, IFR 53, University of Reims 

Champagne-Ardenne, 51 rue Cognacq-Jay, Reims, 51096, France 

2. CReSTIC, University of Reims Champagne-Ardenne, Chaussée du 

Port, Châlons-en-Champagne, 51000, France 

Infrared (IR) micro-spectral imaging is an efficient label-free optical method to analyze biological 

samples and to provide spatially resolved information on the basis of their biochemical 

composition. Recent studies have shown its potential to detect and characterize cancerous tissues 

in their early stages, independently of visual morphology. IR micro-imaging could thus be 

developed as a sensitive, non-destructive and objective diagnostic tool in clinical oncology. The 

discrimination between tumoral and peritumoral tissues relies on the highlighting of subtle IR 

spectral differences. For this, the spectral dataset acquired on biomedical samples is generally 

classified by conventional “hard” clustering algorithms (such as K-means). These clustering 

techniques assign each recorded spectrum to one cluster automatically. K-means of IR data was 

proved to be very efficient to characterize tumoral tissue, but its main drawback is that it cannot 

differentiate the nuances in the assignment of pixels, particularly those located at the interfaces 

between different tissue structures. In this study, we will focus on fuzzy clustering techniques to 

classify data from IR images directly acquired on formalin-fixed paraffin-embedded tissue sections 

of human skin cancers (squamous cell carcinoma and melanoma), without chemical dewaxing. The 

fuzzy clustering extends the traditional clustering concept by allowing each recorded spectrum to 

be assigned to every cluster with an associated membership value. Therefore, for unclear cluster 

boundaries, fuzzy clustering may obtain more reasonable results. However, as with the K-means 

algorithm, fuzzy c-means needs the number of clusters to be pre-specified in advance as an input 

parameter to the algorithm. This algorithm also needs a second input parameter called “fuzziness 

index” which controls the fuzziness of the membership. We developed an algorithm, which can 

generate the optimal number of clusters and optimal “fuzziness index” automatically without a 

priori knowledge about the specificity of the sample. The constructed IR color-coded spectral 

images allow recovering the different histological structures automatically. However, more than 

reproducing classical histology, our algorithm can give access to interesting information on the 

assignment of the IR images pixels to the tissular structures. For each pixel, fuzzy classification 

provides with membership values, permitting to nuance their assignment. Such data are very 

valuable for the pixels located at the interface between tumoral tissue and its microenvironment. 

Thus, heterogeneous transitional areas between tumor and environmental normal tissue were 

identified for the examined tissue sections. These areas cannot be identified on hematoxylin-eosin 

staining or by K-means. Experiments are underway to define the molecular assignments of the 

spectral variations observed in these peritumoral areas. 

PA 105


SERS+SEF study on piroxicam over Ag nanoparticles 

P. SEVILLA 1, 2 , R. DE LLANOS 2 , C. DOMINGO 2 , S. SÁNCHEZ-CORTÉS 2 AND J.V. GARCÍA-RAMOS 2 

1. Departamento de Química Física II, Facultad de Farmacia, 

Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 

28040, Spain. 

2. Instituto de Estructura de la Materia, Consejo Superior de 

Investigaciones Científicas, Serrano 121, Madrid, 28006, Spain. 

Piroxicam (4-hydroxyl-2-methyl-N-2-pyridinyl-2H-1,2,-benzothiazine-3-carboxamide 1,1-dioxide) 

is a nonsteroidal anti-inflamatory drug (NSAID). The <strong>European</strong> Medicines Agency (EMEA 25 June 

2007) has recommended restrictions on the use of piroxicam-containing medicinal products 

because of the risk of gastrointestinal side effects and serious skin reactions. Topical medicines 

containing piroxicam are not concerned by these restrictions. It occurs as a white crystalline solid 

slightly soluble in alcohol and in aqueous solution. It exhibits acid-basic equilibrium with two pKs 

at 1.8 and 5.8. Only the neutral forms of the oxicam drugs are suggested to be medically active. 

Several studies on the behaviour of piroxicam on water solution have appeared recently [1-2]. On 

the other hand metal nanoparticles have become an efficient method to store and deliver drugs in 

sick patients. In this sense, the study of piroxicam in presence of Ag nanoparticles can help to the 

development of new target systems that can avoid the aforementioned side effects and can 

contribute to the understanding of their biological effects. The aim of this work is to characterize 

the piroxicam molecules when they are adsorbed on Ag nanoparticules, using colloids prepared 

through two different methods and at different pHs. We have used metal surface enhanced 

spectroscopy techniques to obtain SERS (surface enhanced raman spectroscopy) and SEF (surfaced 

enhanced fluorescence spectroscopy) spectra. Preliminary results of our work are presented here, 

indicating as well as the molecular characterization the possibility of using SERS as a new method 

for the quantification of the different species of the drug. 

References 

Fig. 1 – Structure of piroxicam 

[1] M.L.S. Silva, M.B.Q. Garcia, J.L.F.C. Lima, E. Barrado, Electroanalysis 19, 1362-1367 (2008). 

[2] M. Gil, A. Douhal, J. Phys. Chem. A 112, 8231-8237 (2008). 

PA 106


Redox dynamics and catalytic activity of Human Sulfite 

Oxidase immobilized on electrodes studied by potential 

controlled Surface Enhanced Raman Spectroscopy 

M. SEZER, R. SPRICIGO, U. WOLLENBERGER, P. HILDEBRANDT, I. M. WEIDINGER 

1. Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 

135, 10623 Berlin Germany 

2. Institut für Biochemie und Biologie, Universität Potsdam, Karl- 

Liebknechtstrasse 24-25, 14476 Golm, Germany 

Human Sulfite Oxidase (HSO) is an enzyme in the mitochondria, which catalyses the oxidation of 

sulfite to sulfate [1]. HSO is present as a dimer where each monomer contains a cytochrome b5 

(heme) and molybdophterin-binding subdomain (Moco domain). Catalytic sulfite oxidation takes 

place at the Moco domain followed by a fast electron transfer to the heme center of the Cyt b5. 

Both domains are connected by a loop region, which provides high flexibility between the 

subdomains. Immobilisation of HSO on metal electrodes makes it possible to control the catalytic 

activity of the enzyme and to use it as an ultrasensitive sulfite biosensor. Electrochemical 

investigations of surface-bound HSO in single-and multilayer systems showed that the catalytic 

activity can be preserved under certain conditions in the immobilized state [2]. Surface enhanced 

resonance Raman (SERR) spectroscopy represents an instructive alternative to electrochemical 

methods since it can selectively probe the active site structure of immobilized proteins, if the 

excitation line coincides with an electronic transition of the protein cofactor (molecular resonance 

enhancement) and the surface plasmon resonance of the metal (surface enhancement). Using the 

potential jump technique time resolved SERR spectra can give detailed information about electron 

transfer kinetic as well as protein dynamics and changes of the cofactor structure during the 

catalytic cycle [3]. We could successfully immobilize HSO on the electrode via electrostatic 

interactions of the negatively charged Cyt b5 subdomain with positively charged self assembled 

monolayers (SAM). Using the 413 nm line of a Kr laser we could monitor the heme in the Cyt b5 

subdomain by SERR spectroscopy. The structure and redox-properties of the heme and the 

catalytic activity of the immobilized enzyme have been studied by time-resolved potential 

controlled SERR spectroscopy. The results are compared with measurements on the isolated Cyt 

b5 domain. 

References 

Fig. 1 – Catalytic mechanism of the immobilized Human Sulfate Oxidase. 

[1] R.M. Garrett, K.V. Rajagopalan, Proc. Natl. Acad. Sci. USA 95, 6394-6398 (1998). 

[2] R, Spricigo, U. Wollenberger, Soft Matter 4, 972 (2008). 

[3] D.H. Murgida, P. Hildebrandt, Chem. Soc Rev. 37, 937-945 (2008). 

PA 107


Molecular Structure and Vibrational Studies of 1, 4naphthoquione 

Using Quantum Mechanical Methods 

P. SINGH , G. SRIVASTAV AND R. A. YADAV 

Dept. of Physics, Banaras Hindu University, India 

Ab initio restricted Hartree-Fock RHF and density functional theory B3LYP calculations were 

carried out to study the molecular structure, atomic charges and Vibrational spectrum of 1, 4- 

naphthoquione (NQ, see figure - 1) molecule. Harmonic forced field and vibrational mode 

calculations provided convinced theoretical evidence for assignments of fundamental vibrational 

mode. This study shows that density functional theory is a powerful approach for understanding 

the vibrational spectra of organic molecules. The calculations predicted a planar structure with C2v 

symmetry for NQ molecule. The quiones and their one electron reduce forms occupy central place 

in electron transfer chemistry and biological energy conversion [1-2]. 

References 

Fig. 1 – The structure of 1, 4 – naphthoquione (NQ) 

[1] A. Crofts, A. C. Wraight, Biochim. Biophys. Acta 726, 149-185 (1983). 

[2] H. H. Robinson, R. A. Crofts, FEBS Lett. 153, 221-226 (1983). 

PA 108


Comparative study of cytosine and its radicals by 

quantum mechanical method 

R. SINGH, S. JAISWAL, M. KUMAR, P. SINGH, G. SRIVASTAV AND R. A. YADAV 

Lasers and Spectroscopy Laboratory, Department of Physics, Banaras 

Hindu University, Varanasi-221005, India 

Cytosine and its derivatives are the compounds of great biological importance because they are 

one of the constituents of nucleic acids [1, 2]. In the present study the vibrational characteristics of 

cytosine and its cationic and anionic radicals have been calculated with the B3LYP level using the 

standard 6-311++G** basis set [3]. It is found that the neutral and the cationic radical have planar 

structures while the anionic radical have non-planar structure with C1 symmetry. In the case of 

anionic radical, both the H atoms of the NH2 group are slightly out of the molecular plane. All H 

atoms carries negative charge and positive charge on the N9 site in the anionic radical irrespective 

of the neutral and cationic radical of cytosine molecule. The C4-N9 bond length is calculated to be 

smaller in the cationic radical of cytosine than the neutral and anionic radical of cytosine. The 

magnitude of the C=O out-of-plane vibrational frequency is calculated to be nearly equal for 

neutral and its anionic radical while increases in its cationic radical by ~ 75 cm -1. The C=O 

stretching frequency is calculated to be highest for the neutral cytosine (1769 cm -1) and almost 

equal for its anionic and cationic radical (1670 cm -1). 

References 

Fig. 1 – Front view of cytosine Molecule 

[1] M. Rozenberg, G. Shoham, I. Reva, R. Fausto, Spectrochim. Acta Part A 60, 463 (2004). 

[2] H.O. Schild, Applied Pharmacology, ELBS, Edinburgh, (1998). 

[3] M. J. Frisch et al., Gaussian 03, Revision C.02, Gaussian, Inc.Wallingford CT (2004). 

PA 109


Low frequency vibration modes from large viruses 

S. SIROTKIN 1 , M. BERGOIN 2 , J. VAN ETTEN 3 AND A. MERMET 1 

1. Université Lyon 1, Villeurbanne, F-69622, France ; CNRS, UMR5620, 

Laboratoire de Physico-Chimie des Matériaux Luminescents, 

Villeurbanne, F-69622, France. sirotkin@pcml.univ-lyon1.fr 

2. Unité de Biologie Intégrative et Virologie des Insectes UMR1231 

INRA - Université Montpellier II 34095 Montpellier Cedex 5, France 

3. Deparment of Plant Pathology, University of Nebraska, Lincoln, 

Nebraska, USA 

From their rigid and compact globular structures, viruses can be considered as “biological 

nanoparticles”. As such, they are expected to feature low frequency vibrational modes that are 

routinely detected from solid nanoparticles [1]. These optical-like modes consist in overall 

oscillatory motions of the nanoparticles on a timescale of the order of the nanosecond to the 

picosecond, depending on the size of the particles. Although long predicted through theoretical 

calculations [2,3] and admittedly conceived as potentially important ingredients of the dynamical 

and elastic behaviors of viruses, these low frequency modes have proved hard pressed to be 

detected experimentally. Our works aim at detecting these modes through low frequency inelastic 

light scattering. Following an investigation dedicated to the search of low frequency vibration 

modes from small (~15-30 nm) plant viruses [4], we have recently focused our interest on larger 

viruses, namely the Chilo Iridescent Virus (CIV) and the Paramecium Bursaria Chlorella Virus 

type-1 (PBCV-1). Both viruses have a diameter of 190 nm and have a well characterized structure. 

We have performed very low frequency Raman/Brillouin measurements on diluted aqueous 

suspensions and highly concentrated pellets of both viruses. The corresponding spectra show a 

broad inelastic feature around 6 GHz, which is q-independent thereby certifying the optical 

character of the detected mode. In order to interpret these data, we have compared the virus 

results to those of polystyrene nanoparticles of identical sizes, in comparable conditions (solutions 

and pellets). This comparison enables the identification of the observed signals from the viruses as 

low frequency modes of their globular structures. 

References 

[1] Duval et al, Phys. Rev. Lett. 56, 2052 (1986), Kuok et al, Phys. Rev. Lett., 90, 25502 (2003) 

[2] H. Ford, Phys. Rev. E 67 (2002), 051924; L. Saviot, D. Murray, A. Mermet, and E. Duval, Phys. Rev. E 67 (2002), 

051924 

[3] F. Tama and C.L. Brooks III, J. Mol. Biol. 318 (2002), 733 

[4] B. Stephanidis, S. Adichtchev, P. Gouet, A. McPherson and A. Mermet, Biophys. J. 93 (2007) 1354 

PA 110


Raman study of biologically active 6-azacytidine and 

related compounds 

I.ALEXEEVA 1 , S. GARASEVYCH 2 , M. IAKHNENKO 2 , L. PALCHYKOVSKA 1 , O. SLOBODYANYUK 2 AND 

I. VASKIVSKYI 2 

1. Institute of Molecular Biology and Genetics, National Academy of 

Science of Ukraine, 150 Zabolotnogo Street, Kyiv 03143, Ukraine 

2. Department of Physics, Taras Shevchenko National University of 

Kyiv, 64, Volodymyrs’ka St., 01033 Kyiv, Ukraine 

Anomalous nucleoside 6-azaC is known as an antimetabolite with wide spectrum of therapeutic 

activities. Reported Raman spectra of anomalous nucleoside 6-azacytidine (6-azaC) were measured 

for the first time. The space group P212121 and perfect quality of 6-azaC microcrystals synthesized 

by us were proved by X-ray characterization. The spectra of the 6-azaC as well as related 

compounds (i.e. cytidine, cytosine, ribose and others) in microcrystalline state and in different 

solutions were measured in range 30-3800 cm -1. Raman peaks positions of the 6-azaC and other 

compounds in the range 500-1800 cm -1 corresponding to intramolecular vibrations remain 

approximately the same when going from microcrystals to solutions (Fig.1). Also spectra of 6azaC, 

cytidine and cytosine have high degree of similarity. It was revealed that some Raman peaks 

of 6-azaC dissolved in D2O have manifested abnormal high frequency shifts with respect to their 

position in H2O solution. Similar results were obtained for 6-azaC microcrystals recrystallized 

from D2O solution as well as for cytidine. To the best of our knowledge this is the first observation 

of abnormal Raman frequency shifts of intramolecular modes under deuteration. In the spectra of 

6-azaC and cytidine calculated with Gaussian 03 package were obtained both normal and 

abnormal shifts close to the measured ones. We assume that abnormal shift occurs due to 

deuteration of intramolecular H-bonds. Details of the abnormal shifts study will be presented at 

the poster session. This work was supported by the Fundamental Researches State Fund of the 

Ministry of Education and Science of Ukraine (grant № F25/137-2008). 

Fig. 1 – Raman spectra of 6-azaC in microcrystalline form (a) and 

in water solutions: D 2O (b) and H 2O (c). 

PA 111


Spectral properties of GFP S65T at cryogenic 

temperatures 

A. SPILOTROS 1 , M. LEVANTINO AND A. CUPANE 



To obtain a consistent interpretation of the temperature dependence of the optical spectra of GFP 

and its mutants both emission and absorption spectra are necessary. Moreover, in the GFP case 

absorbing and emitting chromophores differ: the latter is deprotonated. Hence, to address the 

problem of the nature of the temperature dependence of the optical spectra of GFP one has to start 

with mutants, chromophore of which does not change its protonation state upon the excitation [1]. 

The absorption spectra of GFP mutant S65T were obtained in the 20-290 K temperature range in 

glycerol/water solvent at different pH values. The low temperature spectra revealed two pHdependent 

absorption peaks in the visible range. Varying the pH from acidic (pH 5) to basic (pH 8) 

the equilibrium between these two bands of the spectrum was moved from one to the other to 

study the spectral profile of the bands individually. At intermediate pH values the temperature 

dependence of the spectra gave information about inter-conversion dynamics. At cryogenic 

temperatures the profile of the peaks became narrower and clearly revealed the presence of at least 

three sub bands for each peak. 

References 

320 340 360 380 400 420 440 460 480 500 520 

PA 112 

Abs (O.D.) 

1.0 

0.5 

0.0 

pH6 

20K 

50K 

80K 

110K 

140K 

170K 

200K 

230K 

260K 

290K 

Wavelength (nm) 

Fig. 1 Absorption spectrum of GFP S65T at pH6 in the temperature 

range 20-290 K. 

[1] Solomon S. Stavrov, Kyril M. Solntsev, Laren M. Tolbert, and Dan Huppert, J. Am. Chem. Soc. 128, 1540-1546 

(2006).


Electron impact excitation functions of gas-phase 

adenine molecules 

M.I.SUKHOVIYA, M.O.MARGITICH, M.M.CHAVARHA, I.I.SHAFRANYOSH 

Uzhgorod National University, Uzhgorod, 88000 Ukraine 

lshafr@rambler.ru 

In this paper we report the results of the study of the specific features of the excitation and 

ionization of gas-phase biomolecules by slow electrons. The electron impact excitation and 

ionization of gas-phase thymine molecules is studied in crossed electron and molecular beams. The 

nucleotide base molecules were transported as a molecular beams to the region of their interaction 

with an electron beam. The technique of crossed electron and heterocyclic biomolecular beams 

used in this study has been described in our previous papers [1,2]. The molecular beam is formed 

by an effusion thermal source of a multichannel type and a system of collimating slits. A fiveelectrode 

electron gun with a tungsten cathode was used as an electron beam source. The 

experiments were performed under the following conditions: the electron beam current was 

(0.1-3) .10 --5 A, ∆E1/2~(0.3-0.5)eV (FWHM) energy spread; the concentration of the molecules was 

2 .10 10 cm -3, and the vacuum was at 1 .10 -5 Pa. The emission spectra of adenine were studied in the 

wavelength range 160-600 nm, as well as the electron energy dependences of the effective 

excitation cross sections (excitation functions) of the molecular bands at their peaks. The electron 

beam energy was scanned within 0--200 eV. The molecular emission spectra were obtained and 

the bands are identified. It can be assumed that the nature of the band origin is associated with the 

excitation of the electronic states of both the whole molecule and its nucleotide base nucleotide 

base fragments (dissociative excitation). Note that the spectrum substantially differs from the 

luminescence spectra of the molecules in solutions and polycrystalline films. For example, the 

excitation function for the band at λmax=354 nm has a smooth form and a broad maximum at 80 eV, 

which is characteristic of the excitation of the singlet states. The excitation function for the band at 

435 nm has a maximum located near the threshold, which is characteristic of the excitation of the 

triplet states. The molecular band at 308 nm is the most strong and has the energy threshold 13.7 

eV. The sum of the energy of this spectral transition (4.3 eV) and the ionization energy (9.4 eV) 

amounts to 13.7 eV, which coincides with the excitation energy of this band. Therefore, the upper 

state of this band can be treated as the ionic state of a thymine molecule. Note that the maximum 

of the excitation function of the 286.5nm band is located at the same energy as that of the 

ionization function. 

References 

[1] M.I.Sukhoviya, V.N.Slavik, I.I.Shafranyosh, L.L.Shimon, Biopolim. Klet.,7,77 (1991). 

[2] I.I.Shafranyosh, M.I.Sukhoviya, M.I.Shafranyosh, J.Phys.B;At.Mol.Opt.Phys.39,4155 (2006). 

PA 113


Raman spectroscopy methods for studying supported 

lipid bilayers 

C. S. SWEETENHAM AND I. NOTINGHER 

Nanoscience Group, School of Physics and Astronomy, University of 

Nottingham, University Park, Nottingham, NG7 2RD, UK 

Supported lipid bilayers (SLB) have found wide application as models of cellular membranes in 

biophysical research. SLB consist of amphiphilic molecules that can self-assemble into bilayer 

structures when deposited onto a hydrophilic support from an aqueous solution, either by vesicle 

fusion or the Langmuir-Blodgett technique [1]. Common features of SLB are the presence of 

defects or holes, which can be used to measure their thickness, and phase-separated 

microdomains, which affect the structure and function of membranes and are an important feature 

in many cellular processes. These artificial membranes are well-defined and stable under a variety 

of conditions, allowing characterisation with a broad range of physical methods. Imaging 

techniques such as electron microscopy and atomic force microscopy (AFM) have been used to 

study SLB [2], but these provide purely topographical and mechanical information. Alternatively, 

optical techniques such as Raman spectroscopy can offer a detailed chemical and structural 

analysis of SLB. However the sensitivity and spatial resolution that can be achieved with these 

methods can restrict their effectiveness. Surface-enhanced Raman spectroscopy (SERS) has been 

developed to overcome these limitations, but there is uncertainty concerning the level of signal 

enhancement this technique can provide and whether the localised heating associated with SERS 

may damage SLB [3]. We present a confocal Raman microspectroscopy system optimised for 

studying SLB. This system combines the benefits of Raman spectroscopy with the high spatial 

resolution of confocal microscopy. Furthermore, the additional incorporation of AFM makes it 

possible to directly correlate chemical information with spatial features. We focus on the limits of 

this system for detecting a single SLB and imaging its microdomains, and investigate how SERS 

can be implemented to enhance the signal achieved with confocal Raman microspectroscopy. 

References 

[1] E. T. Castellana, P. S. Cremer, Surface Science Reports 61, 429-444 (2006). 

[2] Y. F. Dufrêne, G. U. Lee, Biochimica et Biophysica Acta 1509, 14-41 (2000). 

[3] E. C. Le Ru, P. G. Etchegoin, Faraday Discussions 132, 63-75 (2006). 

PA 114


Comparison of molecular structure of alkali metal ortho 

substituted benzoates 

R. ŚWISŁOCKA 

Department of Chemistry, Białystok Technical University, Zamenhofa 29, 15-435 Białystok, Poland 

Benzoic acid derivatives represent a wide group of aromatic ligands, which are constituent parts of 

enzymes and other biologically important molecules. The changes in physical, chemical and 

biological properties of them decided about their effect on the biological systems. Benzoic acid and 

its derivatives are commonly used food preservatives as well as antiseptic agents applied in 

various industrial branches: pharmaceutics, textile and cosmetics. The present work is a 

continuation of our recent reports on the vibrational and NMR spectra as well as geometric 

characteristic of benzoic acid derivatives [1-4]. The influence of amino-, nitro-, methoxy-, hydroxy- 

and halogeno substituents in the ortho position towards the carboxylic group as well as alkali 

metal on molecular structure of benzoates were estimated. FT-IR, FT-Raman and NMR spectra of 

the title compounds were recorded and analyzed. Data of chemical shifts in 1H and 13C NMR as 

well as wavenumbers and intensities in IR and Raman spectra of studied benzoate derivatives 

were analyzed in comparison with alkali metal benzoates. Optimized geometrical structures were 

calculated by B3LYP/6-311++G** method. The calculated parameters are compared with 

experimental characteristics of these molecules. 

References 

[1] M. Samsonowicz, R. Świsłocka, E. Regulska, W Lewandowski J. Mol. Struct.837, 220-228 (2008). 

[2] R. Świsłocka, M. Samsonowicz, E. Regulska, W Lewandowski J. Mol. Struct.834-836, 389-398 (2007). 

[3] M. Kalinowska, R. Świsłocka, W Lewandowski J. Mol. Struct.792-793, 130-138 (2006). 


PA 115


Resonance Raman study of sensor domain of 

chemotaxis proteins from G. sulfurreducens 

S. TODOROVIC 1 , M. PESSANHA 1 , Z. GOUVEIA 1 , A. P. FERNANDES 2 , P. R. POKKULURI 3 , Y. Y. 

LONDER 3 , M. SCHIFFER 3 AND C. SALGUEIRO 2 

1. ITQB, Universidade Nova de Lisboa, Av. da Republica EAN, 2780-157 

Oeiras, Portugal 

2. Requimte, Dep. de Química, Faculdade de Ciências e Tecnologia, 

Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 

3. Biosciences Division, Argonne National Laboratory, Argonne, Il 60439, USA. 

Proteins of sensory systems enable bacteria to respond to environmental conditions. Typically, a 

domain in the periplasm senses some external stimulus (CO, NO, O2, metal ions, redox potential 

changes, etc. ), transduces the signal through the cell membrane, subsequently activating the 

cytoplasmic domain. The changes in the latter then generate an intracellular signal that can trigger 

a specific physiological function. Geobacter sulfurreducens, a protobacterium found in subsurface 

soil, possesses an unusually high number of signal transduction proteins. Two out of ten c-type 

heme containing sensor domains were cloned, expressed in E. coli and purified [1]. In order to 

distinguish between their sensing properties and preferences, we undertook detailed resonance 

Raman (RR) spectroscopic study. RR spectra of heme proteins, obtained under Soret band 

excitation, provide information on redox, spin and coordination state of the heme group. 

Component analysis of the RR spectra of soluble domains of chemotaxis proteins from G. s., 

GSU0582 and GSU0935, revealed differences in spin and coordination states of the two proteins in 

the resting states. Moreover, it provided detailed insight into CO and NO binding properties of the 

two proteins in the ferrous for the former, and ferric and ferrous states for the latter ligand. 

References 

1300 1350 1400 1450 1500 1550 1600 

-1 

Raman shift (cm ) 

PA 116 

1300 

1372 

1379 

1592 

1502 

1509 1571 1607 

1408 

1434 

1553 

1644 

1636 

Fig. 1 – Experimental and component RR spectra of ferrous 

GSU0935-NO adduct, showing distribution between 6cLS species 

and 5cHS species with up-shifted frequencies. 

[1] R.R. Pokkuluri, M. Pessanha, Y.Y. Londer, S. J. Wood, N. E. C. H. Duke, R. Wilton, T. Catarino, C. A. Salgueiro, M. 

Schiffer, J. Mol. Biol. 377, 1498-1517 (2008)


PROPERTIES OF RHODOBACTER SPHAEROIDES 

MUTANT I(L177)H REACTION CENTER 

L. G. VASILIEVA, T.Y. FUFINA, R. A. KHATYPOV, V. A. SHUVALOV 

Institute of Basic Biological Problems, RAS, Pushchino, Moscow Region, 

142290 Russia, vasilieva@issp.serpukhov.su, tel: 4967-73-26-80, fax: 4967- 

33-05-33 

The bacterial photosynthetic reaction center (RC) consists of three protein subunits and 10 

cofactors. The RC cofactors are arranged in two symmetrical branches labeled A and B. Each 

branch starts with the primary electron donor P, proceeds via a monomeric bacteriochlorophyll 

(BChl) molecule and a bacteriopheophytin molecule and terminates with a quinine acceptor. The 

efficiency of the photochemical charge separation in RC depends on the properties of electron 

transfer cofactors, their positions and interactions with each other and with the surrounding 

protein environment. Recently new mutant of Rb. sphaeroides was described that had I(L177)H 

substitution in the RC [1]. L177 position is located in the vicinity of BB and PA. Our results show 

that the mutation strongly affects the spectral properties of BChls. The mutant RCs were found to 

be active in charge separation with the quantum yield of P +QA - state formation similar to that in 

the wild-type RC. Em P/P + in I(L177)H RC was shown to be decreased by 50 mV. Pigment analysis shows 

different BChl content in pigment extracts from RCs I(L177)H and the wild type RCs. The acenote - 

methanol (7:2) extract from the wild type RCs contains four BChls and two BPheos per RC and the 

extract from the mutant I(L177)H RCs contains three BChl molecules and two BPheos per RC. It 

was noticed that the color of the denaturated I(L177)H RC protein remained green even after 

multiple steps of pigment extraction by variety of organic solvents. It was estimated that 

approximately one BChl molecule (0.7+ 0.05 ) per one I(L177)H RC remains attached to the RC 

protein. During SDS PAGE the attached BChl moves through the gel along with L-subunit of 

I(L177)H RC indicating covalent binding of the pigment and the mutated protein [2]. Hitherto 

there is no data on a chromophore - protein covalent binding in photosynthetic membrane 

pigment - protein complexes. Putative origin of the pigment-protein covalent binding will be 

discussed. The RC I(L177)H seems to be a promising example for the study of protein-cofactor 

interactions in photosynthetic complexes. 

References 

[1] R.A. Khatypov, L.G. Vasilieva, T.Y. Fufina, T.I. Bolgarina, and V.A. Shuvalov, Biochemistry (Rus) 2005, 70, 1256- 

1261 

[2] Fufina T. Y., Vasilieva L. G., Khatypov R. A., Shkuropatov A. Ya., Shuvalov V. A., FEBS Lett., 2007, 581, 30, 5769- 

5773 

PA 117


Probing conformational changes in G protein-coupled 

receptors using FTIR spectroscopy on azido-labeled 

rhodopsin 

S. YE 1 , T.P.SAKMAR 1 AND R. VOGEL 2 

1. Laboratory of Molecular Biology and Biochemistry, The Rockefeller 

University, 1230 York Ave., New York, NY 10065, USA 

2. Biophysics Group, Institute of Molecular Medicine, University of 

Freiburg, Hermann-Herder-Str. 9, 79104 Freiburg, Germany 

Fourier-transform infrared (FTIR) difference spectroscopy is a powerful experimental tool for 

characterizing changes within a protein on a molecular level, though its application is often 

hampered by congestion and identification of spectral bands and only limited site-specificity of the 

spectral information. Here we report on a conceptual advance of FTIR difference spectroscopy by 

combining it with cutting-edge molecular biology technologies. Amber codon suppression allows 

the site-directed incorporation of an IR-active unnatural amino acide, p-azido-L-phenylalanine 

(azidoF) into the G protein-coupled receptor (GPCR) rhodopsin. The intense anti-symmetric 

stretch vibration of the azido label absorbs at around 2100 cm -1 in a clear spectral window devoid 

of other protein bands and is exceptionally sensitive to the polarity of its surroundings. We show 

the application of this new technique by following movements of single helices of the transmembrane 

receptor in different intermediates of the activation pathway [1], which are controlled 

by the triggering of electrostatic switches in interhelical networks [2]. The combination with sitedirected 

azido labeling considerably expands the capability of FTIR difference spectroscopy by 

adding the site-specific electrostatic information while maintaining its sensitivity to monitoring 

changes of side chains, backbone, and ligands of the whole protein. 

References 

Fig. 1 – The antisymmetric stretch vibration of the azidoF side chain 

absorbs in a clear spectral window and allows to follow helix 

movement during rhodopsin activation and the translocation of the 

azidoF227 side chain from a hydrophobic to a polar environment. 

[1] S. Ye, T. Huber, R. Vogel, T.P. Sakmar, Nature Chem. Biol., in press (2009). 

[2] M. Mahalingam, K. Martinez-Mayorga, M.F. Brown, R. Vogel, Proc. Natl. Acad. Sci. USA 105, 17795-17800 (2008). 

PA 118


Investigation of soil microorganisms by means of 

various spectroscopic methods in combination with 

statistical analysis 

A. WALTER 1 , S. ERDMANN 2 , W. SCHUMACHER 1 , T. BOCKLITZ 1 , M. REINICKE 2 , P. RÖSCH 1 , 

E. KOTHE 2 AND J. POPP 1,3 

1. Dept. of Physical Chemistry, Friedrich Schiller University, 


2. Dept. of Microbiology, Friedrich Schiller University, Neugasse 25, 


3. Institute of Photonic Technology, Albert Einstein Str. 9, 07745 Jena, 

Germany 

Microbial life has conquered highly contaminated environments and developed resistance 

mechanisms, which bear high potential for the development of new bioremediation approaches of 

polluted sites. This requires the isolation and identification of adapted microorganisms and 

analysis of their resistance mechanisms. Raman spectroscopy in combination with chemometrical 

analysis is capable for in vivo investigation and fast identification of microorganisms on molecular 

basis [1, 2]. The filamentous fungi Schizophyllum commune [3] and species of the soil bacteria 

Streptomyces [4] have been isolated from a former disused mining district, located near Ronneburg 

(Eastern Thuringia, Germany). Several Streptomyces strains isolated from the contaminated sites 

exhibiting heavy metal resistances [4]. The characterization, classification and identification of 

different Streptomyces species by various spectroscopic methods were carried out in combination 

with supervised and unsupervised statistics. Within different hyphal parts of S. commune 

mitochondria localization via cytochrome detection by means of Raman spectroscopy in 

combination with k-mean cluster analysis was achieved. The activity of apical versus subapical 

mitochondria was detected by peak analysis of a Raman marker band. 

References 

[1] M. Schmitt, J. Popp, J. Raman Spectrosc. 37, 20-28, (2006). 

[2] K. C. Schuster, I. Reese, E. Urlaub, J. R. Gapes, B. Lendl, Anal. Chem. 72, 5529-5534, (2000). 

[3] E. Kothe, H. Bergmann, G. Buechel, Chem. Erde 65, 7-27, (2005). 

[4] A. Schmidt, G. Haferburg, M. Sineriz, D. Merten, G. Buechel, E. Kothe, Chem. Erde 65, 131-144 (2005). 

PA 119


Continuous-flow step-scan IR spectroscopy for study of 

ligand binding and enzyme catalysis 

C.W.WHARTON 

School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK 

IR spectroscopy has proved useful in the simultaneous determination of chemical processes and 

the associated conformational changes that occur during enzyme catalysis. Rapid scan 

spectroscopy can measure processes in the 10’s of millisecond time range but is limited by 

mechanical factors to this time range or slower. It is often possible to slow down natural processes 

that are faster than this time range by changing the pH or temperature so that they can be brought 

within the range of rapid scan FTIR. However this is not a comfortable process since it can change 

the rate-limiting step of a reaction & one is left with the feeling that the ‘true’ reaction is no longer 

being studied. In particular, it is not possible to measure ligand binding kinetics, which always 

occur on a faster time scale in the low microseconds. There is almost no structural information 

available concerning the detailed dynamics of binding (or dissociation) of substrates to enzymes of 

neurotransmitters to their receptors – an important ‘black hole’ in our knowledge of molecular 

recognition processes. Step-scan IR spectroscopy can access the time range down to nanoseconds, 

and has been applied to bacterio-rhodopsin proton pumping in a most incisive manner. The drawback 

is that it requires the repetitive stimulation of the reaction process many times in order to 

record a spectrum. It works by building up the interferogram step by step, rather than in a 

continuous sweep as in normal FTIR. For processes where the change in absorbance is small a 

large number of kinetic transients must be recorded at each interferometer position. This is fine for 

a photo-stable system such as bacterio-rhodopsin that can be repetitively excited but very difficult 

to achieve for a ‘one-shot’ reaction system since a new sample has to be supplied for every kinetic 

transient at every interferometer position (typically overall a value of several thousand). This 

difficulty could be overcome by using a large number of stopped-flow shots but this would be 

prohibitively expensive in materials. It has been overcome by using continuous flow in a 

microcuvette and by making use of a caged substrate (or neurotransmitter), which is photoactivated 

at each measurement step of the interferogram. I will describe how the method has been 

applied to the study of glutamate binding to its neuro-receptor by Jayaraman [1] and how 

chymotrypsin, transpeptidase and β-lactamase catalyses are being studied using this method. 

Reference 

[1] Q. Cheng, M. Du, G. Ramanoudjame, V. Jayaraman, Nature Chem. Biol. 1, 325-331 (2005) 

PA 120


Ultrafast infrared spectroscopy of isotope labeled 

Riboflavin compounds 

M. M. N. WOLF 1 , R. GROß 1 , T. DOMREATCHEVA 2 AND R. DILLER 1 

1. Fachbereich Physik, TU Kaiserslautern, Kaiserslautern, 67663, 

Deutschland, mawolf@physik.uni-kl.de 

2. Max-Planck Institut für medizinische Forschung, Jahnstraße 29, 

Heidelberg, 69120, Deutschland 

Flavin molecules are well known for their role in biochemistry, as for their enzymatic role in the 

citric acid cycle. Due to their absorption of blue light they are also found as chromophores in blue 

light receptors. This includes the class of Cryptochromes and the BLUF domain of the AppA 

protein, which both bind Flavin-Adenine-Dinucleotide (FAD), as well as the class of phototropins, 

binding Flavin-Mononucleotide (FMN). In our recent work [1] we presented sub-picosecond time 

resolved infrared spectroscopy of Riboflavin in DMSO solution in a wide spectral range from 1100 

cm -1 to 1750 cm -1. The vibrational modes of the electronic ground and first excited states were 

assigned based on quantum chemical calculations. While there was a strong accordance between 

the calculations and the experiment, there were some discrepancies in the carbonyl stretch region, 

possibly due to different hydrogen bonding schemes. In this presentation we will show similar 

measurements together with calculations of two different isotopic labeled Riboflavin 

chromophores – Riboflavin-2- 13C and Riboflavin-4,10a- 13C. With their different position inside the 

flavin ring system the isotopic labels cause distinct couplings resp. downshifts of the carbonyl 

stretch vibrational modes, which eases their assignment. Implications for various intra- and intermolecular 

hydrogen bonding schemes are discussed. 

References 

Fig. 1 – Absorption difference spectra of both isotopic labeled 

Riboflavin (RF) compounds at a delay time of 100 ps after photo 

excitation. 

[1] M. M. N. Wolf, C. Schumann, R. Groß, T. Domratcheva, R. Diller, J. Phys. Chem. B 112 13424-13432 (2008). 

PA 121


New insights in the oxygen-tolerant membrane bound 

[NiFe]-hydrogenase from Ralstonia Eutropha by means 

of 

FTIR and EPR spectroscopy 

I. ZEBGER 1 , M. SAGGU 1 , T. GORIS 2 , S. FRIELINGSDORF 2 , M. LUDWIG 2 , O. LENZ², N. HEIDARY 1 , 

P. HUMMEL 1 , D. MILLO 1 , M. HORCH 1 , M.E. PANDELIA 3 , W. LUBITZ 3 , B. FRIEDRICH², R. BITTL 4 , 

P. HILDEBRANDT 1 AND F. LENDZIAN¹ 

1. Max-Volmer-Laboratory for Biophysical Chemistry, TU Berlin, Straße 

des 17. Juni 135, 10623 Berlin, Germany 

2. Institute of Biology / Microbiology, HU Berlin, Chausseestr. 117, 


3. Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, 

45470 Mülheim/Ruhr, Germany 

4. Institut für Experimentalphysik, WE 1, FU Berlin, Arnimallee 14, 


[NiFe]-hydrogenases catalyze the reversible cleavage of molecular hydrogen [1]. The EPR and 

FTIR study presented here provides the first spectroscopic characterization of the membranebound, 

oxygen-tolerant [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 in its natural 

environment, the cytoplasmic membrane [2]. In addition, we have investigated and the isolated 

hetero-trimeric form of the MBH consisting of the subunits HoxG, HoxK and HoxZ. The large 

subunit HoxG harbors the Ni-Fe active site whereas the small subunit HoxK is dedicated to 

electron transfer via one [3Fe4S] cluster and two [4Fe4S] clusters to the membrane-integral HoxZ 

protein – a cytochrome b which serves as the natural electron acceptor. Initial surface-enhanced 

infrared absorption spectroscopic (SEIRAS) investigations were carried out with a His-tagged 

hetero-dimeric HoxKG form of the MBH [3]. The spectroscopic data reveal a strong similarity of 

the MBH active site with known Ni-Fe centers from strictly anaerobic hydrogenases, like that from 

Desulfovibrio vulgaris Miyazaki F [4]. Most redox states characteristic for anaerobic [NiFe] 

hydrogenases were also identified for the MBH except for one remarkable difference. The 

formation of the oxygen-inhibited Niu-A state was never observed. This observation is directly 

related to the unusual oxygen-tolerance of the enzyme. Furthermore, EPR data showed the 

presence of an additional paramagnetic center at high redox potential (+290 mV), which couples 

magnetically to the [3Fe4S] center in the MBH small subunit. The data indicate a structural and/or 

redox modification at or near the proximal [4Fe4S] cluster. Further insights were derived from 

mutant proteins containing amino acid exchanges close to the proximal Fe-S cluster. The 

spectroscopic results are discussed in light of the oxygen tolerance of the MBH from R. eutropha. 

References 

[1] R. Cammack, M. Frey, R. Robson (2001) Hydrogen As a Fuel: Learning from Nature, Taylor and Francis Ltd., 

London 

[2] M. Saggu, et al. J. Biol. Chem., in press, DOI 10.1074/jbc.M805690200 

[3] N. Wisitruangsakul et. al, Angew. Chem 48, 611-613 (2009). 

[4] C. Fichtner, C. Laurich, E. Bothe, W. Lubitz, Biochemistry 45, 9706-9716 (2006) 

PA 122

Synchrotron and XFEL spectroscopy 13 th ECSBM −−−− Book of Abstracts 

PA 

Synchrotron based X-ray and FTIR micro-spectroscopy 

of myelin 

T. DUČIĆ 1 , S. QUINTES 2 , K.A. NAVE 2 , J. SUSINI 3 , M. RAK 3 AND T. SALDITT 1 

1. Institute for X-Ray Physics, Georg-August University, Friedrich- 

Hund-Platz 1, 37077 Göttingen, Germany 

2. Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Str. 3, 

37075 Göttingen, Germany 

3. <strong>European</strong> Synchrotron Radiation Facility, rue Jules Horowitz 6, 38000 

Grenoble, France 

Myelin is a spiral structure formed by extensions of the plasma membrane of the oligodendrocytes 

in the central nerve system and the Schwann cells in the peripheral nervous system [1, 2]. Each 

myelin sheath segment is separated by spaces where myelin is lacking, the nodes of Ranvier (150– 

200 µm in length), which play a major role in nerve impulse conduction. In this study single 

isolated myelinated neurons were structurally and functionally characterized using synchrotron 

based micro- and nano-beam imaging. In situ analyse of elements and especially trace metals in 

neurons in recent years by synchrotron-based X-ray fluorescence (XRF) achieve significant 

progress. Using the recently developed cryo facility at the <strong>European</strong> Synchrotron Radiation 

Facility (ESRF), we performed element determination in single separated neurons under 

physiological condition. Here we present recent progress in synchrotron X-ray optics to map the 

elemental composition on the subcellular level of myelinated sciatic neuron using highly focussed 

synchrotron radiation for spectro-microscopy and synchrotron based infrared spectroscopy (FTIR), 

in particular close to the node of Ranvier. Mapping of organic compounds, lipids, phosphorus 

components, as well as protein conformation were performed with FTIR microscopy. Our results 

showed high accumulation of phosphorus compounds of the node of Ranvier (Fig. 1.) It is known 

that these parts of neurons are rich in sodium channels [2]; however high concentration and strong 

location of phosphorus compounds was unexpected. After FTIR analysis cAMP and NADPH are 

possible candidates to explain this result. Overall, the high resolution spectroscopy imaging of 

single neuronal cells offers unique information to understand the structural organisation and the 

role of different compounds and especially trace metals in neurochemistry and the process of 

myelinisation. 

Fig. 1 X-ray fluorescence image of sodium (A), phosphorus (B), chloride (C) and sulphur (D) in the single 

isolated axon. Colour scale indicates elements concentration in µg/cm². Image size 50x25 µm, 246x125 

pixels with dwell time 180 ms per pixel. 

References 

[1] A.Peteres J Anat 98, 125–134 (1964). 

[2] T. Boiko, B. Winckler J. Cell Biol. 172:799-801 (2006). 

123


PA 

Protein in solution: analysis of hydrational properties 

and stability by neutron and X-ray scattering techniques 

P. MARIANI, F. SPINOZZI, F. CARSUGHI, M. G. ORTORE AND R. SINIBALDI 

Sezione Scienze Fisiche, Dipartimento SAIFET, Università Politecnica 

delle Marche, Via Ranieri 65, I-60131 Ancona, Italy; e-mail: 

mariani@univpm.it 

Small angle scattering (SAS) of X-rays and neutrons (SAXS and SANS) technique is particularly 

suitable to study structural properties of proteins in solution. With respect to crystallography or 

NMR-based techniques, the main advantage is its capability to investigate all protein species in the 

sample. Processes that can be studied with SAS include shape determination, conformational 

transitions that occur upon binding of effectors or changes in physicochemical conditions, longrange 

protein-protein interaction, aggregation, solvation, folding and unfolding. In the case of 

poly-disperse systems (as during the unfolding process, when the protein conformation is not 

unique) data analysis could be very tricky. However, we have recently shown that a global fit 

strategy, i.e., the simultaneous fit of all the scattering curves based on a suitable physical model, 

can successfully provide relevant structural and thermodynamical information even for mixed 

systems. Three examples will be presented. The first regards the study by SAXS of the unfolding of 

met-myoglobin at pH 4.5 during pressure-induced and pressure-assisted, cold denaturation 

processes. The second concerns the behaviour of β-lactoglobulin in acidic solution as a function of 

the ionic strength, the pressure and the ethylene-glycol co-solvent as studied by SAXS and SANS 

techniques. The last regards a SANS and SAXS study of the solvation properties of model proteins 

(namely, lysozyme and BSA) dissolved in water/glycerol and in water/urea mixtures. In all cases, 

the relevant role that the solvent plays in maintaining the stability of the different proteins has 

been quantitatively described by determining the volume and compressibility changes at 

dissociation or during the unfolding process and by estimating the solvent preferential binding 

coefficients. 

References 

[1] F. Spinozzi, F. Carsughi, P. Mariani, L. Saturni, S. Bernstorff, S. Cinelli, and G. Onori. J. Phys. Chem. B, 111, 

3822-3830 (2007). 

[2] M.G. Ortore, F. Spinozzi, F. Carsughi, P. Mariani, M. Bonetti, and G. Onori. Chem. Phys. Lett., 418, 342-346 

(2006). 

[3] R. Sinibaldi, M. G. Ortore, F. Spinozzi, F. Carsughi, H. Frielinghaus, S. Cinelli, G. Onori, and P. Mariani. J. Chem. 

Phys., 126, 235101(1-9) (2007). 

[4] R. Sinibaldi, M. G. Ortore, F. Spinozzi, S. Funari, J. Teixeira, P. Mariani. <strong>European</strong> Biophys. Journal, 37, 673–681 

(2008). 

[5] F. Spinozzi, M. G. Ortore, R. Sinibaldi, P. Mariani, A. Esposito, S. Cinelli, G. Onori. Journal of Chemical Physics, 

129, 035101(1-9) (2008) 

[6] M. G. Ortore, A. Paciaroni, F. Spinozzi, P. Mariani, H. Amenitsch, J. Ollivier, L. R. S. Barbosa, M. Steinhart, D. 

Russo. The Journal of the Royal Society Interface, in print (2009). 

124


PA 

Theoretical and experimental study of tautomerism in 

guanine and cytosine spectra 

I. L. ZAYTSEVA 1, A. B. TROFIMOV 1 , O. PLEKAN 2 , V. FEYER 2 , K. C. PRINCE 2 , R. RICHTER 2 , 

M. CORENO 3 

1. Quantum Chemistry, Irkutsk State University, 664003 Irkutsk, Russia 

2. Sincrotrone Trieste, in Area Science Park, I-34012 Trieste, Italy 

3. CNR-IMIP, I-00016 Montelibretti (Rome), Italy 

The core level ionization and excitation spectra and the valence shell ionization spectra of guanine 

and cytosine were studied theoretically using the algebraic-diagrammatic construction (ADC) ab 

initio Green's function approach. The calculations were used to interpret the results of recent gasphase 

synchrotron radiation experiments which for the first time allowed for clear observation of 

signals due to various tautomers in the XPS, NEXAFS and valence-shell photoelectron spectra of 

the molecules under study. The Boltzmann population ratios (BPRs) of guanine and cytosine 

tautomers at the experimental temperatures (T = 600 K and 450 K, respectively) were established 

from the results of high-level ab initio thermochemical calculations and were used in theoretical 

modeling. The theoretical spectra obtained are in good agreement with the experimental results, 

allowing for unambiguous assignment of the observed structures in terms of signals from the 

individual atoms and tautomers. In guanine, as well as in cytosine, essentially three tautomers, one 

of which consists of two rotamers, contribute significantly to the resulting spectra. The 

tautomerism is most pronounced in core photoemission spectra, enabling determination of ratios 

of oxo and hydroxy tautomer forms from the experimental data. The experimental tautomer ratios 

agree well with the present theoretical estimates. The core photoabsorption and valence shell 

photoelectron spectra of guanine and cytosine also demonstrate important signatures of 

tautomerism, but most of the bands here are more complex and formed by numerous overlapping 

transitions of different final states and tautomers, so that the observed features are less tautomer 

specific. 

Fig. 1 – Four most stable tautomers of guanine. Experimental (dotted line) and 

theoretical (full lines) O 1s photoionization spectra of guanine showing signals of oxo 

(1, 2) and hydroxy (3, 4) tautomers (shift of theoretical spectrum: -1.0 eV; Gaussian 

broadening: 1.0 eV). 

125


PA 

126


POSTER CONTRIBUTIONS 

SESSION B

Palermo, August 28 – September 2, 2009 13 th ECSBM −−−− Book of Abstracts

Biomedical applications 13 th ECSBM −−−− Book of Abstracts 

Structures and interactions of nucleolipids based 

vectors for gene delivery 

N.ARAZAM 1, 2 , S. KHIATI 3 , F. NALLET 1 , P. BARTHÉLÉMY 3 , B.DESBAT 2 AND L. NAVAILLES 1 

1. Centre de Recherche Paul Pascal, Université Bordeaux1-CNRS, 115 av 

Schweitzer, 33600 Pessac, France 

2. Chimie et Biologie des Membranes et Nanoobjets, Université 

Bordeaux1, UMR 5248 CNRS, ENITAB, 2 rue Robert Escarpit 33600 

Pessac, France 

3. INSERM U869, 146 rue Léo Seignat, 33076 Bordeaux, France 

Gene therapy is a promising tool for developing new strategies against genetic diseases and 

cancers. For medical applications, the vector must be prepared to allow the greatest transfection 

efficiency and the lowest cytotoxicity. Various synthetic vectors designs were used in the past 

years. Recently, DNA delivery systems based on anionic lipids have emerged as a possible 

alternative to cationic lipids [1]. Here, we present a study on anionic nucleolipids [2]. The main 

advantage of those systems is the possibility to modulate the interactions with nucleic acids 

between ionic and base-pairing interactions. We used The Langmuir method coupled to Brewster 

angle microscopy and PMIRRAS infrared spectroscopy to investigate interactions between 

nucleolipids and nucleic acids. We have been able to characterize nucleolipids films and determine 

the different types of interactions according to various physicochemical parameters. We also have 

performed x-ray studies to determine structural properties of nucleolipids-DNA complexes [3]. 

Finally, we have identified a multiphasic organization and the results allow us to establish a 

correlation between the structure of the complexes and their transfection efficiency. 

References 

[1] H. Liang, D. Harries, G.C.L. Wong, Proc. Natl. Acad. Sci. USA 102, 11173-11178 (2005). 

[2] A. Gissot, M. Camplo, M.W. Grinstaff, P. Barthélémy, Org. Biomol. Chem. 6, 1324–1333 (2008). 

[3] S. Khiati, N. Pierre, S. Andriamanarivo, M.W. Grinstaff, N. Arazam, F. Nallet, L. Navailles, P. Barthélémy, 

submitted to bioconjugate chemistry. 

PB 1


The use of the Raman Spectroscopy for the study of 

Biofilm caused by the E. faecalis bacteria in 

periodontitis 

R. AVILA RODRÍGUEZ 1 , A. GONZÁLEZ AMARO 2 , D. RODRÍGUEZ 

OVIEDO 2 , C ARAUJO ANDRADE 3 , FACUNDO RUÍZ 4 , J. R. MARTÍNEZ 4 

1. Programa de Posgrado en Ingeniería Eléctrica, Facultad de Ingeniería, 

Universidad Autónoma de San Luis Potosi. Av. Salvador Nava S/N 

2. Programa de Posgrado en Endodoncia de la Facultad de 

Estomatología de la Universidad Autónoma de San Luis Potosi, Av. 

Salvador Nava S/N 

3. Unidad Académica de Física de la Universidad Autónoma de 

Zacatecas. Zacatecas, Zac 

3. Facultad de Ciencias, Universidad Autónoma de San Luis Potosi, 

78000 San Luis Potosí, S.L.P., México 

In Endodontics the E. faecalis has the hability to invade and to colonize the root canal [1], can 

survive without the support of other bacterium, it has the special capability of building a biofilm to 

protect and to survive, that is why it can resist against the antimicrobials, and begins to infect the 

roots canals [2,3,4,5]. The bacteriological biofilm is a community of bacteria that is in a matrix of 

exopolysaccharides [6, 7] produced by themselves and adhered to a organic or inorganic surface 

to provide protection and more resistance to the external influences, which is why they are 

protected against the action of the antibodies, and protected for the fagocitosis and for the 

antimicrobians treatment. The biofilm can be 1000 times more resistant to the antimicrobians, that 

is way the biofilm is so important to study, to be able to learn what their structure consists of and 

try to make it disappear and to enable the antibiotics to produce their effects. Actually, at this 

time the study of biofilm “in situ“ is limited, but there has been some research with the electronic 

microscopy, and other molecular biology techniques. The Raman spectroscopy is a feasible 

technique to detect the biofilm which has multiple bands due to the biofilm components such as 

carbohydrates, cytocine, amino acids. The advantage of this techique is that it is not necesary to 

prepare the biofilm and goes direct over the sample. 

References 

[1] Love R.M., et al Enterococcus faecalis- A mechanism for its role in endodontic failure, IEJ, 34,399-405,2001. 

[2] Distel J.W. et al., Biofilm Formation in Medical Root Canals, JOE 2002, Vol 28, No. 10 689-693. 

[3] Stuart Charles H., et al., Enterococcus faecalis: Its Role in Root Canal Treatment Failure and Current Concepts in 

Retreatment. 

[4] Chavez de Paz L. Response to alkaline stress by root canal bacteria in biofilms, IEJ, 40 344-355,2007. 

[5] Craig J., et al, Comparrison of the antimicrobial efficacy of 1.3% NaOCl/Biopure MTAD to 5.25% NaOCl/15% EDTA 

for Root canal irrigation, JOE, Vol. 33, No. 1, January 2007 

[6] Biofilms Bacterianos: Cronificación, 2005. 

[7] Lasa I., Biofilms Bacterianos. Actualidad, Vol. 28, Num 2. 2004 

PB 2


Chemometric analysis of biospectroscopic data in R: 

hyperSpec 

C. BELEITES 1 AND V. SERGO 1 

1. CENMAT, Materials and Natural Resources Dept., 

University of Trieste, Via Valerio 6/a, Trieste, I-34127, Italy 

We present a new software package, hyperSpec, that greatly facilitates the analysis of spectral data 

in R [1]. hyperSpec makes R a convenient platform for the analysis of spectral data sets, including 

spectral images and maps. It takes care of data import and export, supplies the means to plot the 

data (spectra, false-color maps, time series, depth profiles, calibration curves) and can be used for 

handling and processing spectra. The actual statistical calculations are performed by functions 

supplied by within the R environment: chemometric methods such as regression, classification and 

cluster analysis are readily available in the statistical software R, as well as the means for 

validation, determination of confidence intervals, etc. In hyperSpec, the spectra can be stored with 

arbitrary amounts of meta-information such as position, sample numbers, constituent 

concentrations, diagnoses, etc. Also, spectral maps/images need neither be rectangular nor evenly 

spaced, and may be combined with spectra without spatial information. Specialized and 

customized methods are needed for the chemometric analysis of biomedical spectroscopic data 

sets (e. g. validation schemes that take into account varying numbers of spectra per patient and 

diagnosis, or robust statistical methods). hyperSpec can easily be extended or used together with 

other R packages.. R is developed with a quality assurance work cycle and participated in a test of 

statistical software [2]. A standardized interface for different data types (classes) and statistical 

methods greatly facilitates the flexible interaction between specialized data (like hyperSpec data 

sets) and specialized data analysis methods. Also interaction with Matlab is easy using R.matlab 

[3]. hyperSpec allows scripting of the data analysis so that computationally intensive calculations 

can be run as batch jobs but there are also functions for user interaction on the plots. Sophisticated 

graphical user interfaces (GUIs) tailored for specific tasks may be built using hyperSpec together 

with other R packages that supply GUI elements. While several software solutions for the 

chemometric analysis of spectral data of biomedical samples exist, none could meet all key 

requirements for the use of spectral data as a diagnostic tool in medicine. Here we present two 

examples of how hyperSpec is used: a cluster analysis of a Raman map of chondrocytes in cartilage, 

and a linear calibration of fluorescence emission of quinine. hyperSpec is hosted at http://r-forge.rproject.org/projects/hyperspec/. 

References 

[1] R Development Core Team, R, A language and environment for statistical computing, Vienna: R Foundation for 

Statistical Computing, ISBN 3-900051-07-0, URL http://www.R-project.org. (2009) 

[2] K. B. Keeling, R. J. Pavur, Computational Statistics & Data Analysis 51, 3811 – 3831 (2007) 

[3] H. Bengtsson, R.matlab - Local and remote Matlab connectivity in R, Mathematical Statistics, Centre for 

Mathematical Sciences, Lund University, Sweden, URL http://www.maths.lth.se/help/R/R.matlab/ (2005) 

PB 3


Infrared imaging: a new tool to refine breast cancer 

prognosis 

A. BÉNARD 1 , C. DESMEDT 2 , V. DURBECQ 2 , G. ROUAS 2 , D. LARSIMONT 3 , C. SOTIRIOU 2 AND 

E. GOORMAGHTIGH 1 

1. Laboratory for the Structure and Function of Biological Membranes, 

Université Libre de Bruxelles, Belgium 

2. Functional Genomics and Translational Research Unit, Department of 

Medical Oncology, Institut J. Bordet, Brussels, Belgium 

3. Department of Pathology, Institut J. Bordet, Brussels, Belgium 

Currently for breast cancer prognosis, clinical guidelines are based on lymph node status, tumor 

size, histological grade, age of the patient, as well expression of various cellular receptors (ER, PgR, 

HER2). However, the existing predictions remain unsatisfactory to identify the best treatment for 

the individual patient. Recently gene expression profiling based on DNA microarrays has brought 

promising results in the field [1]. Even though this approach may help to improve decision-making 

by the oncologist, it remains expensive and does not take into account the cellular heterogeneity of 

the tumor sample. The recently developed IR imaging systems allow the analysis of the different 

components of a tumor, taking into consideration the spatial resolution of the cells. Here, we 

applied for the first time these new IR imaging systems to breast cancer samples. IR spectroscopy 

is based on the absorption of infrared light by vibrational transitions in covalent bonds. While the 

intensities provide quantitative information, the frequencies relate to the nature of these bonds, 

their structure, and their molecular environment. In complex systems such as cells, an infrared 

spectrum is the sum of the contributions arising from proteins, lipids, nucleic acids, and all other 

chemical species present in the cells. IR spectroscopy provides a complete signature which can be 

correlated with the biological properties of the sample. Hundreds of biological applications of this 

technology have been published since it was demonstrated, in the eighties, that the FTIR spectrum 

of bacteria provides a unique fingerprint that allows the identification of bacteria species [2]. Here, 

we analyzed formalin fixed paraffin embedded (FFPE) tissues from breast cancer patients. Good 

quality imaging of the different types of molecules present in the FFPE tissue was obtained on 3 

µm slices. This preliminary study has been made on Tissue Microarrays of invasive breast 

carcinomas. A supervised statistical analysis (Student t-test) was computed between histologic 

grade 1 patients and grade 3 patients. All the significant differences were located in the spectral 

region characteristic of DNA and RNA. A non supervised statistical test (Principal Component 

Analysis) made on this region of the spectra allows the separation of the two groups of patients. It 

is the first principal component (CP1) which represents 80% of the variance that is discriminant. 

We show here in an exploratory study that IR imaging can be applied on FFPE samples, paving the 

way to retrospective studies. It should be possible in a near future to correlate the infrared features 

of the spectra to prognostic parameters. 

References 

[1] C. Sotiriou, C. Desmedt, D. Larsimont, M. Piccart, M. Delorenzi, J. National Cancer Inst. 98 (4) 262-272 (2006) 

[2] D. Naumann, D. Helm, H. Labischinski, Nature 351:81-82 (1991) 

PB 4


Interactions of the beta-blocker drug, propranolol, with 

detergents, cyclodextrins, membranes and living cells studied 

using fluorescence spectroscopy 

R. H. BISBY 1 , S. W. BOTCHWAY 2 , A. G. CRISOSTOMO 1 , J. KAROLIN 3 , A. W. PARKER 2 AND 

L. SCHRÖDER 1 

1. Biomedical Sciences Research Institute, University of Salford, UK 

2. Lasers for Science Facility, Central Laser Facility, STFC, UK 

3. Department of Physics, University of Strathclyde, UK 

Propranolol (I) was developed as a blocker of the beta-adrenergic 

receptor of cells and has been widely used in the treatment of 

hypertension and anxiety. It has previously been noted that 

propranolol is relatively hydrophobic and that its fluorescence 

spectrum in a range of solvents reflects solvent polarity [1], allowing 

membrane interactions to be detected [2]. 

The uptake of propranolol into several cell types, including rat aorta smooth muscle cells, has now 

been imaged using 2-photon (630 nm) excitation of ultraviolet fluorescence (340 nm) using similar 

methods to those described previously for serotonin imaging [3]. The results indicate selective 

uptake of propranolol into acidic organelles, most probably lysosomes, through co-localization 

with Lysotracker® Green DND – 26 dye. Quantitative analysis using fluorescence lifetime 

imaging shows that intracellular concentrations reach levels of several millimolar and are 

sufficient for self quenching of the fluorescence lifetime to be observed. Comparable fluorescence 

lifetimes determined with ultraviolet excitation indicate that spectral changes in solvent systems 

generally are accompanied by shortening of the average excited state fluorescence lifetime and 

development of multiple exponential components, compared with behaviour in neutral aqueous 

solutions where a good single exponential decay with a lifetime of 10.0 ns was measured. In 

contrast, significant changes in the fluorescence spectrum of propranolol on association with the 

hydrophobic environment within sodium dodecyl sulphate (SDS) micelles reflect an increase in the 

fluorescence lifetime to about 18 ns. These results suggest that in the cellular systems, propranolol 

remains strongly associated with the aqueous phase. Further experiments to probe interactions of 

propranolol with amphipathic systems have been undertaken using fluorescence quenching. 

Binding of propranolol to SDS micelles and β-cyclodextrin resulted in substantial decreases in the 

Stern-Volmer constant for steady-state quenching by iodide. This is particularly useful in 

demonstrating association with β-cyclodextrin, where previous experiments based only on 

changes in fluorescence intensity showed a very weak effect [4]. 

References 

[1] C.E.Hunt and R.J.Ansell (2006) Analyst 131: 678-683. 

[2] W.K.Surewicz and W.Leyko (1981) Biochim.Biophys.Acta 643: 387-397. 

[3] S.W.Botchway, A.W.Parker, R.H.Bisby and A.G.Crisostomo (2008) Microsc.Res.Techniq. 71: 267–273. 

[4] A.Y.Glenn, C.A.Fortier, I.V.Jack, X.Zhu and I.M.W|arner (2005) J.Incl.Phenom.Macro. 51: 87-91. 

PB 5 

O NH 2 + 

I


Chemical imaging of articular cartilage 

with Raman mapping 

A. BONIFACIO 1 , C. BELEITES 1, F.VITTUR 2 , E.MARSICH 1 , 

S.SEMERARO 2 , A.FLAMIGNI 2 , S.PAOLETTI 2 AND V. SERGO 1 

1. Dept. of Materials and Natural Resources, University of Trieste, Via Valerio 6a, Trieste, 34100, Italy 

2. Dept. of Life Sciences, University of Trieste, Via Giorgeri 1, Trieste, 34100, Italy 

Cartilage is considered one of the simplest tissues in the body, consisting of one type of cells (i.e. 

chondrocytes) and an extracellular matrix whose main components are collagen, proteoglycans 

and water [1]. Despite its simple structure, the processes involved in arthritis, a widespread 

pathology, are still largely unknown. In the last decade, the need for tools to characterize this 

tissue at molecular level led to the application of several imaging techniques to articular cartilage. 

In particular, FT-IR showed to be a valuable tool to characterize this tissue [2]. We applied another 

imaging technique based on vibrational spectroscopy, i.e. Raman mapping [3], to obtain detailed 

information about the distribution of different chemical species in articular cartilage. The images 

obtained with Raman mapping show the distribution of the different cartilage constituents with a 

lateral resolution of 1 micron. With this resolution, details about the sub-cellular structure of 

chondrocytes have been resolved, and the distribution of the tissue components in the matrix 

immediately adjacent to cells is depicted with unprecedented detail. Moreover, by using the 

hyperSpec software package [4], multivariate data analysis has been applied to cartilage Raman 

maps, yielding additional information otherwise difficult to extract from the univariate analysis 

employed in the previous FT-IR studies on this tissue. These results show the potential of Raman 

mapping for the detailed characterization of articular cartilage, and open the way to further 

studies on the differences between healthy and arthritic tissue at molecular level. 

Fig. 1 – (from left to right): bright field image of a cartilage tissue section of 100x80 microns together 

with false color images obtained by multivariate analysis (e.g. PCA, HCA) showing the relative distribution 

of several chemical species; the average spectrum for the whole section is shown on the right. 

References 

[1] Serafini-Fracassini A. and Smith J.W. (1974), Churchill Livingstone, London 

[2] Boskey A., Camacho N.P. (2007) Biomaterials, 28, 2465-2478 

[3] Krafft C. and Sergo V. (2006) Spectroscopy, 20 (5-6), 195-218 

[4] C.Beleites and V.Sergo, J.Stat.Soft., in preparation (see http://r-forge.r-project.org/projects/hyperspec) 

PB 6


Development of biosensor based on hyperpolarized 

Xenon-129 for NMR and MRI 

A. STOPIN 1 , T. BROTIN 1 , J.P. DUTASTA 1 

C. BOUTIN 2 , Y. BOULARD 2 , F. LETEURTRE 2 , A. SANSON 2 , N. JAMIN 2 

G. HUBER 3 , H. DESVAUX 3 , M. CARRIERE 3 , P. BERTHAULT 3 

1. CNRS, Ecole Normale Supérieure de Lyon, Laboratoire de Chimie, 46 

Allée d’Italie, F-69364 Lyon, France 

2. CEA,iBiTecS, 91191 Gif-sur-Yvette, France 

3. CEA, IRAMIS, SIS2M et URA CEA/CNRS 331, Laboratoire Structure 

et Dynamique par Résonance Magnétique, 91191 Gif-sur-Yvette, France 

Nuclear magnetic resonance is widely used to get spectroscopic and imaging information on 

biological samples. One of the main challenges is to develop biomarkers as contrast agents, of high 

sensitivity and relevant for biomedical application. Hyperpolarized Xenon-129 appears to be an 

interesting candidate because of a) its biocompatibility and solubility in biological tissues, b) the 

enhancement of its NMR signal as a result of the hyperpolarization, which should allow the 

detection of low metabolite or receptor concentrations. c) its response to the local environment 

leading to a wide chemical shift bandwidth [1]. In a recent approach, it has been shown that xenon 

can be directed to given biological targets through encapsulation in functionalized cage-molecules 

[2, 3]. In this purpose, some 129Xe-NMR based biosensors have been built from cryptophane cores, 

hosts known to interact strongly with xenon [4, 5]. These cryptophanes are tethered with ligands of 

interest [6] (Fig. 1). Targeting the transferring receptor highly expressed at cell surface is used as a 

model to evaluate and develop the technique. The first in vitro data using hyperpolarized 129Xe 

have been obtained on transferring-cryptophane biosensor in the presence of K562 cells. These 

results are promising for future work on MRI techniques using small animal models. 

References 

Fig. 1 – schematic representation of the transferrin-cryptophane biosensor 

[1] B.M. Goodson, Using injectable carriers of laser-polarized noble gases for enhancing NMR, MRI, Conc. Magn. Reson 

11, 203- 223 (1999). 

[2] M. M. Spence, S. M. Rubin, I. E. Dimitrov, E. J. Ruiz, D. E. Wemmer, A. Pines, S. Qin Yao, F. Tian and P. G. Schultz 

Proc. Natl. Acad. Sci. USA 98, 10654-10657 (2001) 

[3] P. Berthault, G. Huber and H. Desvaux Prog. NMR Spectrosc. 55, 35-60 (2009). 

[4] G. Huber, L. Beguin, H. Desvaux, T. Brotin, HA. Fogarty, JP. Dutasta, P. Berthault, J Phys Chem A. 112, 11363-72 

(2008). 

[5] Thierry Brotin, and Jean-Pierre Dutasta Chem. Rev. 109, 88-130 (2009). 

[6] V. Roy, T. Brotin, J.-P. Dutasta, M.-H. Charles, T. Delair, F. Mallet, G. Huber, H. Desvaux, Y. Boulard and 

P.Berthault ChemPhysChem, 8, 2082-2085 (2007). 

PB 7


APOMORPHINE VIBRATIONAL STRUCTURE REVEALED BY RAMAN AND SERS 

SPECTROSCOPIES 

M. Casella (1,3, ��), A. Lucotti (2), M. Tommasini (2), F. Gramatica (1), E. Di Fabrizio (3,4) 

and G. Zerbi (2) 

(1) Lab. Biofisica e Nanomedicina, Polo Tecnologico, Fondazione Don Gnocchi IRCCS-ONLUS, 

Via Capecelatro 66, 20148 Milan, Italy 

(2) Dip. di Chimica, Materiali e Ingegneria Chimica “G. Natta”, Politecnico di Milano, 

Piazza L. Da Vinci 32, I-20133 Milan, Italy 

(3) BIONEM Lab, Dip. di Medicina Sperimentale e Clinica, Università Magna Graecia, 

viale Europa, 88100 Catanzaro, Italy 

(4) INFM-TASC (National Institute for the Physics of Matter)- S.S.14 km 163,5 in Area Science Park, 

34012 Basovizza-Trieste, Italy 

� Corresponding Author: mcasella@dongnocchi.it 

Apomorphine (APO - aporphine-10,11-diol, CA Registry Number 58004) is a not narcotic 

derivative of morphine discovered in 1869 by Mattheisen and Wright [1]. It is a well-known strong 

short-acting dopamine agonist at D1 and D2 dopamine receptors, used in the treatment of patients 

with advanced Parkinson’s Disease (PD). 

Its inherent instability complicates its application in clinical practice. In fact, aqueous solutions of 

apomorphine undergo spontaneous oxidative decomposition turning green in the presence of light 

and air [2]. Thus, because of its unfavourable pharmacokinetic properties (it is usually required a 

constant level of APO in the blood during PD treatment) and its chemical instability, monitoring of 

the haematic drug level by means of laser spectroscopy methods (e.g. Raman spectroscopies based 

methods) would represent a turning point for the PD treatment planning. 

In order to understand the vibrational spectrum of this compound, apomorphine hydrochloride and 

its commercial drug formulation (Apofin ® ) were examined by means of Raman and SERS 

spectroscopies. 

In this work Raman and SERS spectra of apomorphine (both salt and commercial drug) and 

oxidation products of apomorphine (acqueous solution) are reported. 

Moreover, the vibrational structure of Apomorphine and Oxoapomorphine have been calculated by 

means of density functional theory (DFT), thus allowing to assign the main peaks observed in the 

experimental Raman and SERS spectra. 

The spectroscopic features identified are fingerprints of the molecule and constitute the basis of 

further experimental works aimed to the development of suitable biomedical SERS sensors. 

References 

[1] A. Mattheisen et al, Proc. R. Soc. Lond. B. Biol.Sci. 17, 455 (1869) 

[2] J. M. P. J. Garrido et al, J. Chem. Soc. Perkin Trans. 2, 1713 (2002) 

PB 8


Effect of different anticancer drugs on prostate cancer 

PC-3 cells classified by imaging FTIR. 

A. DERENNE 1 , R. GASPER 1 , A. BÉNARD 1 AND E. GOORMAGHTIGH 1 

1. Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and 

Bioinformatics, Université Libre de Bruxelles (ULB) 

The number of anticancer agents that fail in the clinic far outweighs those considered effective, 

suggesting that the selection procedure for progression of drug molecules into the clinic requires 

improvement. Traditionally, new drugs are evaluated for their potential to kill cancer cell lines. 

This approach is obviously not sufficient, and molecules with new modes of action are required. 

We suggest here that the infrared spectrum of cells exposed to anticancer drugs could offer an 

opportunity to obtain a fingerprint the metabolic changes induced by the drugs. Because the 

infrared spectrum of cells yields a precise image of all the chemical bonds present in the sample, 

different drug actions are likely to each yield a unique fingerprint characteristic of the “mode of 

action” of the therapeutical agent under investigation. In turn, drug-induced metabolic disorders 

should be amenable to classification similar to the ways in which bacteria gender, species, and 

strains can be classified [1]. Our laboratory has already shown IR spectroscopy can be used to 

evidence metabolic changes induced by one drug on a cell line as a function of time [2]. In the 

present communication we examine human prostate cancer PC-3 cell line exposed to 8 well 

described antimitotics. In a first step the IC50 at 72 hrs incubation time were determined. For FTIR 

imaging, PC-3 cells were exposed to the IC50 concentration of each drug for 48 hrs. Cells were then 

harvested, washed in an isotonic solution and deposited on a BaF2 window. IR spectroscopic 

images were recorded using a Bruker FTIR spectrometer equinox coupled to a Hyperion 3000 

imaging system equipped with a mercury cadmium telluride (MCT)-based focal plane array (FPA) 

detector of 64×64 pixels (Bruker Optik, Ettlingen, Germany). Images of 4096 IR spectra at 8 cm -1 

spectral resolution were acquired by coadding 256 interferograms. All the data processing was 

carried out by the program “Kinetics” running under Matlab. Using principal component analysis 

(PCA), it was found that after 48 hrs of incubation, cells treated with drugs inducing DNA or 

metabolic alterations could be distinguished from the non-treated ones while spectra arising from 

cells blocked in metaphase by the drug were confined in a delineated space among the non treated 

cells. We show with Student test performed between each drug and the non treated cell spectra 

that the different molecules tested induced different spectral modifications. Furthermore, drugs 

known to induce the similar type of metabolic disturbances appeared to cluster when the spectrum 

shapes were analyzed. Finally, supervised statistical methods (PLS) were used to point out spectral 

modifications specific of each molecule and each drug family. Taken all together these data 

suggest that FTIR could be used for the classification of drug action. 

References 

[1] Naumann,D., D.Helm, and H.Labischinski. Nature 351 (1991) 81-82. 

[2] Gasper,R., J.Dewelle, R.Kiss, T.Mijatovic, and E.Goormaghtigh. Biochim.Biophys.Acta 1788 (2009) 1263-1270. 

PB 9


PB 

FT-Raman and SERS characterization of self-assembling 

oligopeptides 

M. DI FOGGIA 1 , C. FAGNANO 1 , P. TADDEI 1 , A. TORREGGIANI 2 , 

M. DETTIN 3 , S. SANCHEZ-CORTES 4 , A. TINTI 1 

1. Dept. of Biochemistry “G. Moruzzi”, University of Bologna, Via Belmeloro 8/2 , Bologna, I-40126, Italy 

2. ISOF-CNR, Via P. Gobetti 101, Bologna, I-40129, Italy 

3. Dept. of Chemical Process of Engineering , University of Padova, Via Marzolo 9, Padova, I-35131, Italy 

4. IEM-CSIC, Serrano 121, Madrid, E-28006, Spain 

The interest in functionalised biomimetic materials used in prosthetic applications as bone 

substitutes has led to studies on regular alternating polar/non-polar oligopeptides such as EAK-16 

(AEAEAKAK)2, first synthesised by Zhang et al. [1]. These peptides have a preferential beta-sheet 

structure, are resistant to proteolytic cleavage and able to self-assemble into an insoluble 

macroscopic membrane under physiological conditions. Their ability to create such stable 

structures derive from the hydrophobic interactions between the aliphatic groups of non-ionic 

residues and complementary ionic bonds between acidic and basic amino acids: this stability can 

be enhanced by the regulation of pH and the presence of monovalent ions. In this context, we 

studied different oligopeptides derived from EAK-16, but modified in their sequence by amino 

acid substitution or by adding at the N-terminus of the sequence the RGD motif. The latter is 

present in the integrins located in the bone extracellular matrix and is able to control osteoblast 

adhesion. Raman spectroscopy was used to investigate the influence of the modifications in the 

sequences on the self-assembly capability of the peptides. In particular, useful qualitative and 

quantitative information on the secondary structure and the hydrogen bonding strength were 

provided by the different amide stretching modes [2]. A SERS study in solution was performed in 

order to understand the interaction way of the EAK-16 derivatives and a titanium oxide rough 

surface: similar interactions are supposed to take place when oligopeptides interact with the 

surface of prosthetic devices. Peptides resulted to interact with silver colloids through their ionic 

moieties, in particular the carboxylate ions of Glutamate and Aspartate residues (as revealed by 

the enhancement of the COO - symmetric stretching band) as well as by the amino groups of Lysine 

residues. The insertion of Tyrosine as spacer did not alter the peptide secondary structure but gave 

a very interesting SERS spectrum, indicating the establishment of the metal-surface interaction 

through the ionic moieties of the peptide, as well as the aromatic ring of Tyrosine residues. 

References 

[1] S. G. Zhang, T. Holmes, C. Lockshin, A. Rich, Proc. Natl. Acad. Sci. USA 90, 3334-3338 (1993). 

[2] A. Tinti, M. Di Foggia, P. Taddei, A. Torreggiani, M. Dettin, C. Fagnano, J. Raman Spectr. 39, 250-259 (2008). 

10


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Unsupervised tissue characterization of a rabbit liver Vx2 

carcinoma model using infrared imaging 

H. D’INCA 1 , M. WASSEF 2 , J. NAMUR 1 , F. PASCALE 3 , E. LY 1 , 

M. MANFAIT 1 AND A. LAURENT 4 

1. MEDyC UMR CNRS 6237, Reims, France 

2. APHP- Lariboisiere Hospital, Dept of Pathology, Paris, France 

3. CR2i APHP- INRA, Jouy-En-Josas, France 

4. APHP- Lariboisiere Hospital, Dept of Interventional Neuroradiology, Paris, France 

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer. The difficulties 

posed by HCC management have prompted the development of numerous potential curative 

treatments such as drug eluting implants or percutaneous injection of materials that kill cancer 

cells. As such, an animal model of liver tumour is very important in the search for an efficient 

therapeutic method. The rabbit Vx2 tumor is a fast-growing carcinoma model, which is being used 

commonly to study different aspects of tumour behaviour under these new therapeutic 

approaches [1]. But it requires analytical techniques which can detect and quantify the tissue 

changes induced by these treatments. FTIR spectral imaging (FTIR SI) of histology sections has 

been recently described as an accurate tool for characterization of tumor tissue [2]. This technique 

has the advantage to be automated and unsupervised. Our aim was to apply FTIR SI for the 

discrimination of the different tissular areas of rabbit liver Vx2 model (tumoral area, necrosis and 

intact liver) in view of their quantification. Nine rabbit livers were injected with Vx2 carcinoma cell 

preparation. Tumor bearing livers were resected 14 days after, formalin fixed and paraffin 

embedded. Two adjacent sections (10µm) were cut from each sample: one stained with 

hematoxylin-eosine (H&E) and one analyzed with FTIR SI. Spectral data were processed by 

multivariate analysis. Firstly, spectral images were computed by K-means in 5 clusters and were 

then correlated with classical H&E histology for validation of clusters assignment. Secondly, a 

predictive model based on these clusters was created using linear discriminant analysis (LDA). 

LDA model was validated by leave-one-sample-out cross-validation method [3] and by correlation 

with H&E histology. K-means classification correlated strongly with H&E images for the three 

types of tissue: normal liver parenchyma (93.5%), viable tumor (93.2%) and intratumoral necrosis 

(98.5%). The sensitivity and specificity of the LDA model was 97.6% and 98% respectively for 

normal liver parenchyma, 96.9% and 94.2% for viable tumor, and 88.4% and 98.8% for intratumoral 

necrosis. The surface of necrotic tissue represented 35±13% of the tumor area as predicted by the 

LDA model, which is in good agreement with the observations on radiological images. FTIR SI 

coupled with LDA modelling allows non-supervised characterization and quantification of tissular 

lesion in Vx2 liver tumor with a good accuracy. Perspectives are to apply FTIR SI on treated 

samples by drug eluting implants. 

References 

[1] EM. Mostafa, S. Ganguli, S. Faintuch, P. Mertyna, SN. Goldberg, J Vasc Inter Radiol 19, 1740-1748 (2008). 

[2] RK. Sahu, S. Mordechai, Future Oncol 1, 635-647 (2005). 

[3] C. Beleites, R. Salzer, Anal Bioanal Chem 390, 1261–1271 (2008). 

11


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Vibrational characterization of birch bark extract, 

betulin anti-cancer agent 

S. PINZARU 1 , A. FALAMAS 1 AND C. DEHELEAN 2 

1. Dept. of Physics, Babes Bolyai University, Kogalniceanu 1, RO 400084, Cluj-Napoca, Romania 

2. Victor Babeş University of Medicine and Pharmacy, Faculty of Pharmacy, Eftimie Murgu Square 2, RO- 

300041, Timişoara, România 

Most of the pentacyclic triterpenes species from extract possess an extremely important 

antitumour activity, with a low toxicity [1, 2]. Therefore, their structure-function relationship is of 

highly importance in the field. In this respect, the Fourier transform- Raman (FT-Raman) and FT- 

IR spectra of birch bark natural extract from Betula Pendula Roth were analyzed with the intention 

of gaining a deeper insight into the structure and vibrational spectra of betulin and of further 

suggesting the possibility of cyclodextrin complexation [3]. Betulin (lup-20(29)-ene-3β,28-diol), 

found mainly as crystalline deposits in the outer layer of the bark, can be extracted by sublimation 

or extraction using organic solvents, as proved from our earlier study [4], in which the best solvent 

was found to be dichloromethane [3]. The promising biological activity of this compound has led 

us to perform detailed spectroscopic and theoretical DFT investigation on its molecular structure. 

The theoretical spectra of betulin resemble well the experimental ones, acquired using a Bruker 

spectrometer as shown in the Fig. 1. The most intense bands in the Raman spectrum were 1195 cm - 

1(assigned to mixed CH bending, CH3 rocking and C-CH3 stretching), 1440 cm -1 (attributed to CH3 

and CH2 bending), 1645 cm -1 (assigned to CH2-CH3 bending and H2C-CH3 stretching) and the very 

strong band at 2927 cm -1 (assigned to CH, CH2 and CH3 stretching). To achieve a precise 

assignment of Raman bands observed experimentally and for a detailed understanding of the 

optimized geometry of the molecular structure, we employed density functional calculations. First 

of all, a geometry optimization calculus was employed using Gaussian 03 software package at the 

B3LYP/6-31G(d) level of theory. Afterwards, a frequency theoretical calculus was performed using 

the same basis set, in order to predict the IR and Raman spectra of the molecule. No negative 

frequency modes were obtained, proving that a true minimum on the potential energy surface was 

found. 

References 

[1] C. A. Dehelean, C. Tatu, S. Cîntă Pânzaru, C. Peev, C. Soica. G. Tanasie, S. Anghel, Toxicology Letters, 164, 1, 

(2006) S228. 

[2] S. C. Pinzaru, N. Leopold, W. Kiefer, “Vibrational spectroscopy of betulinic acid HIV inhibitor and of its birch bark 

natural source”, Talanta, 57, 2002, 625-631. 

[3] C. M. Soica, C. A. Dehelean, C. I. Peev, G. Coneac, A. T. Gruia, “ Complexation with HPGCD of some pentacyclic 

triterpenes. Characterization of their binary products”, Farmacia, vol. LVI, 2, 2008, 182-190. 

[4] C. A. Dehelean, S. Cîntă Pînzaru, C. I. Peev, C. Soica, D. S. Antal Characterization of birch tree leaves, buds and 

bark dry extracts with antitumor activity”, J Optoelectr. Adv. Mat. 9, 3, 2007, 783-787. 

12


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SERS prospect of different organs from mouse and rat 

specimens exposed to UV radiation 

A. FALAMAS 1, 3 , S. CINTA PINZARU 1 , C. DEHELEAN 2 , CH. KRAFFT 3 AND J. POPP 3,4 

1. Babes Bolyai University, Dept. of Physics, Kogalniceanu 1, RO 400084, Cluj-Napoca, România 

2. Victor Babeş University of Medicine and Pharmacy, Faculty of Pharmacy, Eftimie Murgu Square 2, RO- 

300041, Timişoara, România 

3. Institute of Photonic Technology, Albert-Einstein-Straße 9, 07745, Jena, Germany 

4. Institute of Physical Chemistry, Helmholtzweg 4, 07743, Jena, Germany 

This study is trying to evaluate the capacity of Raman and surface enhanced Raman spectroscopy 

(SERS) to differentiate organs and to detect the structure and conformation of molecular 

components from healthy and cancerous tissues. Since most diseases are induced by biochemical 

processes including mutation and/or infection, they are accompanied by molecular composition 

changes in the tissues. Raman spectroscopy provides detailed information about the biomolecular 

composition of tissues, which might be used to distinguish between normal and malignant ones. In 

this study Raman spectra from liver, lung, skin and tumor tissues originating from two types of 

mice species, C57 and NMRI and one type of rat, Spraghe Dawley respectively, were acquired. 

Several specimens were previously exposed to UV-B radiation and were treated with oral DMBA 

(dimethyl 1,2 benzanthrazen) solution, known to induce cancer in different organs. In the end the 

subjects presented skin injuries in a variety of ways, including both acute and chronic responses. 

Cryosections were prepared from the samples collected from organs immersed in formalin 

solution mixed with colloidal silver and single point Raman spectra were acquired. The Raman 

signatures of normal lung and liver tissue samples from mice and rats were characterized in vitro. 

Lung spectra exhibited characteristic peaks which were attributed to CH2 bending vibrations of 

lipids and CH2 and CH3 bending vibrations of proteins, amino acid side chain vibrations and 

amide I band of proteins. These bands can also be seen in liver spectra, though there are slight 

differences in intensity and wavenumbers. The spectra of the skin samples, show vibrations of 

water, proteins and lipids. The Raman spectra of normal skin are dominated by collagen, but also 

bands from lipids, DNA and the amide III and I bands. Raman images were collected to identify 

regions showing SERS effect and FTIR imaging was also applied as a complementary technique. 

First results will be presented. 

References 

[1] Dehelean C, Soica C, Peev C. et al. Pentacyclic triterpenes interventions in skin pathology/toxicity and treatment: 

in vitro and in vivo correlations, Buletin USAMV-CN, 65, 2008. 

[2] A.Lorincz, D.Haddad, et al., Raman spectroscopy for neoplastic tissue differentiation: a pilot study, Journal of 

Pediatric Surgery, 39, 6, 953-956. 

[3] Barry B.W, Edwards H.G.M., Williams A. C., Fourier Transform Raman and Infrared vibrational study of human 

skin: Assignment of spectral bands, J. Raman Spectroscopy, 23, 641-645, 1992. 

[4] Rachel E. Kast, Gulay K. Serhatkulu, et al., Raman Spectroscopy Can Differentiate Malignant Tumors from Normal 

Breast Tissue and Detect Early Neoplastic Changes in a Mouse Model, Biopolymers, 89, 235-241, 2007. 

13


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CD spectroscopy as a tool in studying the interaction 

between metal ions and antitumor compounds 

M.M.L. FIALLO 1 , P. SHARROCK, 1 V. BRUMAS, 1 AND C. METHENITIS 2 

1. LERISM LU 48, Paul Sabatier University Toulouse 3, F-31062, Toulouse, France 

2. Inorg. Chem. Lab., Dept. Chemistry, University of Athens, 15771 Panepistimiopolis, Athens, Greece 

Metal ions are involved in the biological mechanisms of antitumor compounds, such as bleomycin 

[1] and streptogrin [2]. More recently it has been shown that the biological activity of a derivative 

of CC-1065 could be tuned by the interaction of metal ions [3]. However, for other antitumor 

compounds, such as anthracycline, mitomycin C (MMC) or altromycin B (AltroB), the role of metal 

ions on its antitumor properties remains unclear. Our interest in the biological effects of metal 

complexes of antitumor compounds prompted us to investigate if MMC, its derivative 2,7diamino-1-hydroxymitosene 

(MEC) and ALB form stable complexes. As a part of a project aimed 

at the characterization on the interaction between antitumor compounds and metal ions by CD 

spectroscopy, in a first step we assigned the electronic transitions of the chromophores of MMC, 

MEC and AltroB to the specific bands in their CD spectra, as a function of the pH. In a second part, 

this phenomenological approach was applied in studying the spectroscopic modifications 

occurring in the interactions of these antitumor compounds with metal ions, such as copper(II), 

palladium(II), platinum(II) and gold(III). By using CD spectroscopy we observed that MMC forms 

a stable complex with palladium(II) [4], whereas MEC can bind either copper(II), palladium(II), 

and gold(III). For the latter ligand, the binding mode of the metal ion is related to the conformation 

assumed by the organic molecule. In the case of AltroB, CD spectroscopy allows to identify the 

solution structure of its complexes with copper(II), palladium(II), and platinum(II) [5]. 

References 

[1] Y. Sugiura, T. Takita, H. Umezawa, Met. Ions Biol. Syst. 19, 81-108 (1985). 

[2] J. Hadju, Met. Ions Biol. Syst 19, 53-80 (1985). 

[3] D.L. Boger, S.E. Wolkenberg, C.W. Boyce, J. Am. Chem. Soc. 122, 6325-6326 (2000). 

[3] M.M.L. Fiallo, E. Deydier, M. Bracci, A. Garnier-Suillerot, K. Halvorsen, J. Med. Chem. 46, 1683-1689 (2003). 

[5] N. Nikolis, C. Methenitis, G. Pneumatikakis, M.M.L. Fiallo, J. Inorg. Biochem. 89,131-141 (2002). 

14


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Study of porcine cutaneous wound healing 

by Fourier transform infrared microspectroscopy 

P. FLOCH 1, J. NAMUR 1, M. WASSEF 2, H. D’INCA 1, M. MANFAIT 1, A. LAURENT 3,4 

1. MEDyC UMR CNRS 6237, Reims, France 

2. APHP- Lariboisiere Hospital, Dept of Pathology, Paris, France 

3. CR2i APHP- INRA, Jouy-En-Josas, France 

4. APHP- Lariboisiere Hospital, Dept of Interventional Neuroradiology, Paris, France 

Cutaneous wound healing is a complex phenomenon with considerable medical applications. The 

aim of this study is to show the potential of Fourier transform infrared microspectroscopy (FTIR- 

MS) to assess quantitatively the molecular changes induced by cutaneous wound healing. We 

developed an animal model consisting of small punch biopsies in the back skin of pigs. Skin 

samples were then obtained at different times and analysed in histology with common stainings, 

for classical morphological description and quantification. The distribution of collagen, the key 

component of skin wound healing, could be assessed using Red Sirius stain. Additionally, FTIR- 

MS was performed on thin tissue sections (8µm) of pig skin obtained at different time points after 

punching (4, 8, 15 and 29 days) to look for quantitative biochemical information about several 

elements simultaneously. FTIR images were obtained on 21 different biopsies with a Perkin Elmer 

Spectrum Spotlight 300 FTIR Imaging system using the “image” mode and a 25µm/pixel 

resolution. Characteristic spectra of each skin type tissue (epidermis/dermis/scar tissue), localized 

with a control HES stained slide, were extracted from each infrared image. Spectra were compared 

statistically using univariate non parametric (Kruskal-Wallis KW) and multivariate (Hierarchical 

Cluster Analysis HCA) analysis. HCA could differentiate between scar tissue, epidermis and 

dermis at each time of wound healing. Scar tissue was associated with epidermis for time points 

between 4 and 15 days and with dermis at 29 days. Peak assignment showed that the three types of 

tissue were differentiated on collagen specific bands (1035 cm -1, 1204 cm -1, 1280 cm -1, 1330cm -1) [1]. 

The absorbance on collagen peaks increased significantly with time in scar tissue (p


PB 

Conception and realization of new Biosensors based on 

FTIR-ATR devices 

1 A.GOLDSZTEIN, 2 M.VOUÉ, 2 J.DE CONINCK, 2 J.CONTI, 3 J.MARCHAND-BRYNAERT, 3 S.DEVOUGE, 

4 P.TULKENS, 4 S.CARYN, 4 K.BETHOIN, 1 F.HOMBLÉ , 1 E.GOORMAGHTIGH. 

1. Laboratory for the Structure and Fonction of Biological Membranes, 

Center for Structural Biology and Bioinformatics. Free University of 

Brussels (ULB), Belgium 

2. Center of Molecular Modelisation Research. University of Mons- 

Hainaut, Belium. 

3. Laboratory of Organic and Medicinal Chemistry. Catholic University 

of Leuven, Belgium. 

4. Unit of Cellular and Molecular Pharmacology. Catholic University of 

Leuven, Belgium 

Biosensors are devices based on the specific recognition of an analyte of interest by a target [1-2]. 

The biodetection process is based on the transformation of the interaction into an electrical, optical 

or other signal. Biosensors have already attracted intensive interest in many different fields such as 

medical diagnostics and control, environmental analysis and monitoring of biotechnological 

processes. Different surface sensitive techniques can be applied to detect the ligand-receptor 

interactions depending of the nature of the sensor support [3-4]. We are developing a new device 

based on FTIR spectroscopy which is suitable for the investigation of ligand-receptor interactions. 

The new biosensor chip is constituted of an attenuated total internal reflection (ATR) germanium 

element, transparent in the infrared, whose surface has been modified to obtain a covalent binding 

of a receptor [5]. The infrared response is close to zero until the ligand binds in the vicinity of the 

interface . The functionalized surfaces have already been successfully used for detection of 

proteins or of small molecules (biotin, ATP) [6]. In the present communication we demonstrate the 

validity of the approach for measuring the binding of antibiotic molecules. We show that 

vancomycine is easily detected when binding onto a D-Ala D-Ala peptide immobilized on the 

surface of the sensor. The major advantage of the present device, called BIA-ATR sensor, is that it 

provides the user with the entire IR spectrum of the analyte, i.e it allows the accurate identification 

of the nature of the analyte binding onto the receptor. Another advantage is it sensitivity. Small 

antibiotics, short peptides or protein phosphorylation are easily picked up. 

References 

[1] Leech, D. Chem. Soc. ReV. 1994, 23, 205-213. 

[2] Andreescu, S.; Sadik, O. A. Pure Appl. Chem. 2004, 76, 861-878. 

[3] Lo¨fas, S.; Malmqvist, M.; Ro¨nnberg, I.; Stenberg, E.; Liedberg, B.;Lunsdtro¨m, I. Sens. Actuators B 1991, 5, 

79-84. 

[4] Malmqvist, M. Nature (London) 1993, 361, 186-187. 

[5] Devouge, S.; Salvagnini, C.; Marchand-Brynaert, J. Bioorg. Med. Chem. Lett. 2005, 15, 3252-3256. 

[6] Goormaghtigh, E.; Raussens, V.; Ruysschaert, J. M. Biochim. Biophys. Acta 1999, 1422, 105-185. 

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Multivariate analysis of FTIR and Raman spectra of 

lactobacilli isolated from kefir grains 

P. MOBILI 1 , C. ARAUJO-ANDRADE 2 , A. LONDERO 1 , C. FRAUSTO- 

REYES 3 , J. R. MARTÍNEZ-MENDOZA 4 , R. IVANOV-TZONCHEV 2 , 

G. DE ANTONI 1 AND A. GÓMEZ-ZAVAGLIA 1 

1. Centro de Investigación y Desarrollo en Criotecnología de Alimentos, La Plata, Argentina 

2. Unidad Académica de Física de la Universidad Autónoma de Zacatecas, México 

3. Centro de Investigaciones en Óptica, A.C, Aguascalientes, Ags. México 

4. Facultad de Ciencias, UASLP, San Luis Potosí, S.L.P. México 

Modern technical developments have made Raman and FTIR spectroscopy fast, powerful and 

easy-to-use analytical techniques, combining detailed spectral fingerprints, non-destructivity and 

minimal sample preparation. Over the years, both methodologies have been developed and 

utilized for microbiological applications. Several research groups have already worked out on the 

identification procedures for bacteria using FTIR and Raman spectroscopies [1]. For these 

purposes, spectroscopic information is considered as mathematical data on which chemometric 

techniques, such as Principal Component Analysis (PCA), Partial Least Square-Discriminant 

Analysis (PLS-DA) among others [2] are applied, allowing discrimination and classification of 

bacteria at the species and even at the strain level [3]. In this work, a combined approach of 

vibrational spectroscopic methodologies and multivariate analysis was used for the 

characterization of three species of heterofermentative lactobacilli isolated from kefir grains: L. 

kefir, L. parakefir and L. brevis [4,5]. The microorganisms were cultured and harvested in the 

stationary phase and further liophylized for the registration of the FTIR and Raman spectra. A 

second derivative algorithm was used to enhance and differentiate the FTIR features. The Raman 

spectra could be analysed without any further treatment. Cluster analysis enabled strains to be 

grouped according to their spectral diversity. Using principal component analysis (PCA), L. kefir 

and non-L. kefir strains could be clearly differentiated in the principal component space when the 

1700-1500 cm -1 Raman range was analysed. No clear discrimination was found when the FTIR 

second derivative spectra were used for the analysis. To develop a classification rule, partial least 

squares discriminant analysis (PLS-DA) was carried out. This method allowed the discrimination 

and classification of the sixteen strains under study in two groups: L. kefir and non- L. kefir. The 

prediction model was better for the FTIR data. The novelty of this work resides from one side, in 

the use of two complementary methodologies (Raman and FTIR spectroscopy) for the 

discrimination of heterofermentative species isolated from kefir. From the other side, multivariate 

analysis was used for the first time in the classification and discrimination of L. kefir from the non- 

L. kefir ones. 

References 

[1] H A. Bosch, M. A. Golowczyc, A. G. Abraham, G. L. Garrote, G. L. De Antoni, O. Yantorno, Int. J. Food Microbiol. 

111, 280-287 (2006). 

[2] M H. Martens, T. Næs, “Methods for calibration”, in Multivariate Calibration, Chapter 3, Wiley, Chichester, England, 

(1989). 

[3] C. Mello, D. Ribeiro, F. Novaes, R. J. Popi, Anal. Bioanal. Chem. 383, 701-706 (2005). 

[4] P. Mobili, A. Londero, T. Roseiro, E. Eusebio, G. De Antoni, R. Fausto, A. Gómez-Zavaglia, Vibrat. Spectrosc. doi: 

10.1016/j.vibspec.2008.07.016. (2008). 

[5] P. Mobili, C. Araujo-Andrade, A. Londero, C. Frausto Reyes, E. A. Araiza-Reyna, F. Ruiz, J. R. Martínez-Mendoza, 

G. De Antoni, A. Gómez-Zavaglia, Vibrat. Spectrosc. submitted VIBSPEC-D-09-00041. 

17


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Influence of growth conditions on hydrophobicity of 

Lactobacillus acidophilus LA5 and Bifidobacterium 

lactis Bb12 cells and characteristics of FT-IR spectra 

L. SHAKIROVA, L. AUZINA, P. ZIKMANIS, M. GAVARE AND M. GRUBE 

Institute of Microbiology & Biotechnology, University of Latvia, 

Kronvalda boulevard 4, LV-1010, Riga, Latvia 

Lactic acid bacteria, such as Lactobacillus acidophilus and Bifidobacterium lactis are of considerable 

technological and commercial importance because of their role in the manufacturing and 

preservation of many fermented food products, but they also have probiotic properties and shown 

beneficial effect on health. We have found, that the values of cell surface hydrophobicity (CSH) of 

L. acidophilus LA5 and B. lactis Bb12 cells change in response to varied growth conditions (phase of 

growth, concentration or type of carbon source, presence of oxygen). An evaluation of FT-IR 

spectra (Vertex, HTS-XT, cluster and quantitative analyses) obtained for cells convectively dried 

under mild (≤ 50°C) conditions revealed substantial changes of chemical composition depending 

on the CSH level of L. acidophilus LA5 and B. lactis Bb12. A decrease of the carbohydrate level was 

observed in proportion to increased CSH values of both cultures alongside with the elevated 

protein content of more hydrophobic cells. Similar relationships we have detected and recently 

reported by bacteria Zymomonas mobilis 113S [1]. The results of present study could help to specify 

the appropriate physiological state of L. acidophilus LA5 and B. lactis Bb12 to perform their 

probiotic properties. 

References 

[1] L. Shakirova, L. Auzina, M. Grube, P. Zikmanis, J. Ind. Microbiol. Biotechnol. 35, 1175-1180 (2008). 

18


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Application of FT-IR for characterization of biomass 

isolated from drinking water 

K. TIHOMIROVA 1 , M. GRUBE 2 AND T. JUHNA 1 

1. Dept. of Water Engineering and Technology, Riga Technical 

University, Azenes street 16/20-263, Riga, LV 1048, Latvia 

2. Institute of Microbiology and Biotechnology, University of Latvia, 

Kronvalda blvd. 4, Riga, LV 1586, Latvia. 

Drinking water companies are responsible for producing and supplying safe, high quality water 

for costumers. The drinking water quality should meet the standard at the customer tap, therefore 

the supplier is forced to analyze the water quality not only at the outlet from the treatment plant 

but in the distribution network as well. The aim of this study was identification and discrimination 

of microorganisms in water samples from different points of distribution network in Riga, Latvia. 

The classification of bacteria is generally based on the morphology and biochemical reactions of 

the bacteria. These measurements are time consuming, requiring training and expertise. Infrared 

(IR) spectroscopic measurements of bacteria followed by a formal chemometrics analysis could 

offer advantages of speed and consistency [1]. Since the amount of pathogenic micro-organisms in 

water is low, after one or more purification processes, concentration is necessary to detect and 

quantify these organisms. Simmons [2] describes a cross flow ultra filter (Hemoflow-filter) that is 

used for the concentration of microorganisms in water. An important advantage of this filter is that 

it concentrates parasitic protozoa, bacteria, spores and viruses/phages. This study was performed 

on drinking water from drinking water plants from Riga, used as water sources surface water and 

groundwater. Here we present results of concentrated drinking water samples which were 

investigated by Fourier Transform Infrared (FT-IR) spectroscopy. Presence of all typical peaks of 

main biochemical cell components: lipids – 2928 – 2856 cm -1 (CH2, CH3 simetric/asimetric 

stretching vibrations); proteins - Amide I and II – 1549 and 1655 cm -1 ; nucleic acids – 1242 cm -1 

(P=O), and carbohydrates - 1080 cm -1 (C-O-C, C-O, C-O-H, P=O of PO2 -, valent stretching 

vibrations of COC groups and ring vibration modes in the composition of cyclic structures) 

verified biomass while qualitative and quantitative differences of components indicated various 

strains or their mix/consortium. Results of both analytical methods showed that water samples 

contain significant amount of biomass. We found distinct difference in the FT-IR spectra of 

biomass isolated from treated groundwater or surface water. In some Hemoflow samples 

Fluorescence in Situ Hybridization (FISH) method indicated E. coli while Pseudomonas fluorescens 

was identified in all samples. This study showed that combination of biomass separation and FT- 

IR spectral analysis is a rapid and simple approach for characterization of microorganisms in 

drinking water. 

References 

[1] D. Lefier, B. Beccard, M. Bradley, “Classification of bacteria using FT-IR”, Thermo Fisher Scientific, USA, 

AN51396_E 04/07M (2007). 

[2] Simmons III, D. Otto, M. D. Sobsey, C. D. Heaney, F. W. Shaefer III, D. S. Francy, “Concentration and detection 

of Cryptosporidium oocysts in surface water samples by method 1622 usinf filtration and capsule filtration”, 

Applied and Environmental Microbiology, 67(3), 1123-1127 (2001). 

19


PB 

FT-Raman spectroscopic study of thoracic aortic wall 

subjected to uniaxial stress 

M. GĄSIOR-GŁOGOWSKA 3 , M. KOMOROWSKA 3 , J. HANUZA, 1 

M. MĄCZKA 2 , M. KOBIELARZ, 4 R. BĘDZIŃSKI, 4 S. SZOTEK, 4 

K.MAKSYMOWICZ, 5 AND K. HERMANOWICZ 2 

1. Department of Bioorganic Chemistry, Wrocław University of Economics, Komandorska 118, 53-345 

Wroclaw, Poland 

2. Institute of Low Temperature and Structure Research, Polish Academy 

of Sciences, P.O.Box 1410, 50-950 Wrocław 2, Poland 

3. Institute of Biomedical Engineering and Instrumentation Wroclaw University of Technology, WybrzeŜe 

Wyspiańskiego 27, Wrocław 50-370, Poland 

4. Division of Biomedical Engineering, Experimental Mechanics, Institute of Machine Design and Operation, 

Wroclaw University of Technology, WybrzeŜe Wyspiańskiego 27, Wrocław 50-370, Poland, 

5. Department of Forensic Medicine, Medical Faculty, Wrocław Medical University, Borowska 213, 50-556 

Wrocław, Poland 

The combination of FT-Raman spectroscopy and uniaxial tensile tests (MTS Synergie 100 testing 

machine) was used to probe the microstructural changes in secondary protein structure of an 

aortic wall, when subjected to different stress levels. The analysis in controlled strain clearly 

highlights different tresholds of tension for the harvested material cut in two directions: 

circumferential and longitudinal and in agreement with the macroscopic mechanical analyses. 

Application of strain in the circumferential direction does not lead to any noticeable changes in 

bandwidths of the Raman bands. When sample is strained in longitudinal direction, some Raman 

bands become narrower and the bands situated at similar wavenumbers are better resolved. 

Stress-controlled Raman’s band analysis shows that modes: 938 cm -1 assigned as (Cα-C) of α-helix, 

1660 cm -1 - amide I (unordered structure of elastin) and 1668 cm -1 amide I (collagen triple helix) 

undergo a wavenumber shifting, althought the modes 1004 cm -1 assigned as a phenyl ring 

breathing mode and 2940 cm -1 δ(CH3), ν(CH2) are not affected during the elastic behaviour (0 – 

35%). Explicit correlation were found between the Raman's band shifting and the level of 

mechanical stress, involving participation of elastin alone transmitting low stresses in the fibers 

direction (circumferential) and of both elastin and collagen (longitudinal) in transmitting 

physiologic and high stresses. 

20


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A conformational study of usnic acid 

M.P.M. MARQUES 1 , J.B.P. DA SILVA 2 , C.S.M. MENDES 2 , 

J. TOMKINSON 3 AND L.A.E. BATISTA DE CARVALHO 1 

1. “Molecular Physical-Chemistry”, Univ. Coimbra, Portugal 

2. Fundamental Chem. Dep., Fed.Univ. Pernambuco, Brazil 

3. ISIS Facility, The Rutherford Appleton Laboratory, Chilton, Didcot, OX11 0QX, United Kingdom 

Usnic acid is a naturally occurring polyphenolic derivative (2,6-diacetyl-1,2,3,9b-tetrahydro-7,9dihydroxy-8,9b-dimethyldibenzofuran-1,3-dione) 

found in lichen [1]. It is considered a potentially 

valuable therapeutic agent, in view of its wide range of pharmacological properties [2-4], such as 

antibiotic, antibacterial, antiviral, anti-malarial, anti-inflammatory, analgesic, antioxidant and 

anticancer. Therefore, interest in this compound has grown since the 1990´s, partly due to the 

increasing resistance to the traditional antibiotics developed by patients, which has led to a search 

for novel therapeutic alternatives (e.g. phytochemicals and other non-synthetic drugs). Since usnic 

acid occurs in distinct tautomeric forms (Fig. 1), its activity being dependent on the species present, 

it is of the utmost importance to determine which one predominates at physiological conditions. A 

complete conformational analysis is presently reported, through a combination of quantum 

mechanical (DFT) calculations and vibrational spectroscopy (Raman, FTIR and INS), aiming at a 

detailed elucidation of the structural preferences of the compound, including its intra- 

/intermolecular H-bonding pattern and the keto-enol tautomerism. Apart from the solid state 

analysis, spectra were also obtained for solutions, with solvents and distinct concentrations, in 

view of determining the effect of the environment on the compound´s conformational behaviour. 

Combination between the complementary data yielded by the optical Raman/FTIR techniques, the 

INS experiments and the calculations, enables a complete assignement of the vibrational spectra of 

usnic acid, as well as an understanding of its H-bonding motif, both in condensed phase and in 

solution. This allows to identify the predominant tautomer in physiological medium, i.e. the major 

species interacting with the compound´s biological receptor(s) and hence responsible for its 

pharmacological activity. 

Fig. 1 – Most stable structures calculated (B3LYP/6-31G**) for the keto (A) and enol (B) tautomers of 

usnic acid. 

References 

A B 

[1] B. Dixon, Lancet Infectious Diseases 5, 534 (2005) 

[2] M. Cocchietto et al. Naturwissenschaften, 89, 137 (2002) 

[3] I. Francolini et al. Antimicrobial Agents and Chemotherapy, 48, 4360 (2004) 

[4] E. Fernández et al. Photochem.Photobiol., 84, 1065 (2006) 

21


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Study of heparin-protamine complexation by means of 

fluorescence spectroscopy, light scattering and 

analytical ultracentrifugation 

J. MAURER, V. VOGEL, W. MÄNTELE 

Institute of Biophysics, Johann Wolfgang Goethe-University 

Frankfurt/Main, Max von Laue-Str. 1, 60438 Frankfurt a. M., Germany 

The administration of heparin, a linear glycosaminoglycan, has been used clinically for many years 

as the standard procedure for anticoagulation treatment [1]. Particularly in surgery, heparin is 

applied in very high doses. But so far, there is no method available for a precise determination of 

the heparin concentration (activity). Often, an additional dose, partly in the range of the already 

applied amount, has to be given because the degradation of heparin varies individually. These 

problems do not assure a precise blood coagulation management for the individual patient and 

frequently lead either to internal bleeding or local coagulations. Here we present two different 

methods for the direct determination of heparin in human blood plasma. Both methods use the 

specific binding between heparin and protamine. Protamine is a small, highly positive charged 

polypeptide, which is clinically used to neutralize the activity of heparin [2]. Both multi-angle light 

scattering technique and analytical ultracentrifugation were used for characterisation of heparinprotamine 

complex size distribution. The observed radius of the heparin-protamine complexes 

was in the range between 10 nm and 200 nm and the complexation process was completed after a 

time between 5 min and 15 min. Both parameters depend on the type of solution in which the 

experiments were carried out (physiological saline solution, model HSA solution and blood 

plasma). The first method for the determination of heparin in human blood plasma which is 

presented here is based on our multi-angle light scattering technique and uses the fact that 

protamine and heparin form nanoscale particles which can be observed through an increased 

intensity of the scattered light [3, 4]. This intensity is proportional to the number of the heparinprotamine 

particles and therefore gives direct evidence to the heparin concentration being present 

in the solution. This system is currently being tested in different clinical centres. The second 

method is a fluorescent spectroscopic technique that also uses the complex formation between 

heparin and protamine. Protamine was labelled with a fluorescent dye and the change of its 

fluorescence characteristics upon binding with heparin was monitored. The intensity of that 

change gives information about the existent heparin concentration. This method is very sensitive 

and can therefore complement the light scattering technique especially for low heparin 

concentrations. Both techniques will be combined to a reliable, quick, sensitive and inexpensive 

clinical heparin test which will help to improve blood coagulation management. 

References 

[1]: Harenberg, J., Fenyvesi, T., Hämostaseologie 2004; 24 (4); 261-278. 

[2]: Chargaff E, Olson KB, Journal of Biological Chemistry 1937; 122 (1); 153-167. 

[3]: Vogel, V.; Häse, C.; Baykut, D.; Mäntele, W.; Zeitschrift für Herz-, Thorax- und Gefäßchirurgie 2007, 21 (4); 140- 

147. 

[4]: GIT SPEZIAL SEPARATION 01/2007, 25-27, GIT VERLAG GmbH & Co. KG, Darmstadt (http://www.biophys.unifrankfurt.de/layout/pdf/2007-GIT-Separation-Heparin.pdf) 

22


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Theoretical DFT modeling of the Raman spectra of 

platinum anti-cancer drugs: picoplatin (AMD473) 

R. WYSOKINSKI, K. HELIOS AND D. MICHALSKA 

Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze 

Wyspianskiego 27, 50-370 Wroclaw, Poland 

Picoplatin, AMD473, cis-[PtCl2(NH3)(2-picoline)] is a new generation platinum complex designed 

to overcome platinum resistance associated with the chemotherapy of cancer. It shows a 

remarkable activity superior to cisplatin and carboplatin in a variety of solid tumors. Picoplatin is 

currently under the clinical phase III trial in SCLC (small cell lung cancer) and phase II trial in 

metastatic colorectal and prostate cancers [1]. Raman spectroscopy is a very useful tool for study 

the binding of platinum drugs to DNA in aqueous solution, because water is a weak Raman 

scatterer, and the bands corresponding to Pt-ligand vibrations are quite strong in the Raman 

spectra. In our earlier studies [2,3] we have shown that the theoretical Raman spectra of cisplatin 

and carboplatin calculated by the modified mPW1PW91 density functional method show very 

good agreement with experiment. In this work we present the molecular structures and the 

Raman spectra predicted for picoplatin, two transient species formed during its hydrolysis, and for 

its complex with model DNA base (9-methylguanine). The unequivocal assignments of the 

platinum-ligand vibrations in the spectra have been made on the basis of normal coordinate 

analysis. The presented results can greatly facilitate the identification of transient species, which 

are formed during the stepwise hydrolysis, and the formation of the DNA-picoplatin adduct, by 

using Raman spectroscopy. 

References 

[1] J. R. Eckardt, D. L. Bentsion, O.N. Lipatov, J. Clin. Oncol. 27, 2046-2051 (2009). 

[2] R. Wysokinski, J. Kuduk-Jaworska, D. Michalska, J. Mol. Structure (Theochem) 758, 169-179 (2006). 

[3] D. Michalska, R. Wysokinski, Chem. Phys. Lett. 403, 211-217 (2005). 

23


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Vibrational study of portland cement-derived materials 

for dental applications 

E. MODENA 1 , P.TADDEI 1 , A. TINTI 1 , M.G. GANDOLFI 2,3 , P.L. ROSSI 2 AND C. PRATI 3 

1. Biochemistry Department, University of Bologna, Via Belmeloro 8/2, Bologna, I-40126, Italy 

2. Department of Odontostomatological Sciences, Endodontic Clinical Section, Laboratory of 

Biomaterials and Oral Pathology, University of Bologna, Via S. Vitale 59, Bologna, I-40126, Italy 

3. Department of Earth Sciences, University of Bologna, Piazza di Porta S. Donato 1, Bologna, I- 

40126, Italy 

Calcium silicate cements have been recently developed as root-end filling materials. They are 

hydraulic materials able to set in the presence of moisture, allowing their use in oral surgery and 

in all conditions where blood and fluid contamination hampers the correct use of other materials. 

Calcium silicate cements, such as mineral trioxide aggregate (MTA) and white portland cements, 

have shown satisfactory biological properties; moreover, they may induce remineralisation of 

partially demineralised dentine. Our study was aimed at comparatively investigating the in vitro 

bioactivity of three calcium-silicate cements derived from modified white portland cement: wTC 

(white tetrasilicate cement), FTC (wTC added with fluoride because of its mitogenic effect on 

osteoblasts) and Pro-Root MTA, a commercial cement used as reference. Bismuth oxide was 

included in all cements as radioopacifier. To elucidate the influence of the fluoride doping agent 

on bioactivity, the cements were aged for different times (from 1 to 28 days), at 37°C, in a 

phosphate-containing physiological solution, i.e. Dulbecco’s Phosphate buffered saline, DPBS. 

ATR/FT-IR and micro-Raman spectroscopy were used to investigate the presence of deposits on 

the surface of the cements and the composition changes of the cement as a function of the ageing 

time. Vibrational spectroscopy allowed to identify the phases present in the unhydrated cement 

powders (alite, belite, gypsum, anhydrite, calcium carbonate) as well as their different hydration 

rates (alite > belite; anhydrite > gypsum). Interestingly, the portlandite phase was found only in 

the interior of the cements, indicating its release into the storage medium, which consequently 

increased its pH. After one day of ageing, all the cements showed the presence of a carbonated 

apatite deposit; both IR and Raman spectroscopy revealed that this deposit was meanly thicker 

and more homogeneous on FTC. From a quantitative point of view, the Raman I960(Apatite)/I311(Bismuth 

oxide) intensity ratio was identified as marker of the deposit thickness. This ratio attained meanly 

higher values for FTC especially at short ageing times. At increasing ageing time the maturation of 

the apatite phase proceeded, as revealed by the progressive decrease in intensity of the Raman 

band at about 1000 cm -1 (attributable to the HPO4 2- ion). In the light of these results and of the 

ability to support cell growth, attachment and cell-surface interactions, these cements appear 

promising as endodontic sealers and root-end filling materials. 

24


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In vivo elution properties of doxorubicin loaded beads 

revealed by microspectroscopies 

J. NAMUR 1 , M. WASSEF 2 , S. CITRON 3 , A. LEWIS 4 , JM. MILLOT 1 , 

M. MANFAIT 1 , A. LAURENT 5,6 

1. MEDyC UMR CNRS 6237, Reims, France, 

2. Pathology, Lariboisiere Hospital, Paris France 

3. Radiology, Piedmont Hospital, Atlanta, GA, United States 

4. Biocompatibles UK Ltd, Farnham, United Kingdom 

5. Neuroradiology, Lariboisiere Hospital, Paris, France. 

6. Matière et Systèmes Complexes UMR CNRS 7057, Paris, France. 

Drug eluting beads (DEB) are polymeric calibrated microspheres measuring 100µm to 300µm in 

diameter, and loaded with the anticancer molecule doxorubicin (DOXO) [1]. They are used in the 

treatment of liver cancers [2,3] with the idea that, compared to intravenous chemotherapy, they 

can increase the local dwell time and concentration of drug inside the tumor. While DOXO-DEB 

have demonstrated they can provide a sustained release of drug for at several months in vitro [4], 

their elution properties have never been assessed in vivo. We aimed to apply Fourier transform 

infrared microspectroscopy (FTIR-MS) and microspectrofluorimetry on tissue sections of DOXO- 

DEB treated livers to determine in situ 1) the amount of drug still retained inside the DEB and 2) 

the distribution and the concentration of the drug in the tissue around the beads. In a pig liver 

model without any tumor, we first evidenced that DOXO-DEB can release their drug load in vivo 

for a period of at least 3 months after the injection and that the drug may diffuse up to a distance 

of 600µm from the bead. In liver tumors explanted from 6 patients after DOXO-DEB injection, we 

further confirmed that DOXO-DEB can provide levels of drug above cytotoxic threshold (>1µM) 

[5], with a maximum delivery in the first hours after implantation. High concentrations of DOXO 

are associated with necrosis of the tissue further supporting the efficacy of the drug eluting system 

in the killing of tumor cells. Microspectroscopies are a very valuable tool to evaluate in vivo 

elution properties of drug eluting systems. 

Fig. 1 – DOXO concentration profiles in the 

tissue around DEB in liver tumors resected 

at different time points after DOXO-DEB 

treatment. Dot line : cytotoxic level [5]. 

References 

[1] A. L. Lewis, M. W. Gonzalez, A. W. Loyd et al. J. Vasc. Interv. Radiol. 17, 335-342 (2006) 

[2] M. Varela, M. I. Real, M. Burrel et al. J. Hepatol. 46 (3), 474-481 (2007) 

[3] K. Malagari, K. Chatzimichael, E. Alexopoulou. Cardiovasc. Intervent. Radiol. 31 (2), 269-280 (2008) 

[4] M. V. Gonzalez MV, Y. Tang, G. J. Phillips et al. J. Mater. Sci. Mater. Med. 19, 767-775 (2008) 

[5] J. J. Chuu, J. M. Liu, M. H. Tsou et al. J. Biomed. Sci.14 (2), 233-244 (2007) 

25


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Investigating Phenotypic Microbial Heterogeneity by 

Infrared and Confocal Raman Microspectroscopy 

DIETER NAUMANN, PETER LASCH AND ANTJE HERMELINK 

Robert Koch-Institute, Berlin Germany 

A whole plethora of techniques has been used to get insight into the development, structure and 

composition of various complex multi-cellular microbial communities like colonies, fruiting bodies 

or biofilms. We have developed vibrational spectroscopic techniques to investigate the phenotypic 

heterogeneity of microbial growth in liquid cultures and macro-colonies, which can be adapted 

also to the analysis of more complex multi -cellular communities such as fruiting bodies, biofilms, 

or colonies growing under natural conditions. Macro-colonies from Legionella, Bacillus, and Candida 

strains were chosen as model systems of multi-cellular communities to evaluate the technique. We 

used an infrared micro-spectroscopic (IR-MSP) approach that implicates excision of the intact 

macro-colonies together with the underlying agar, freezing and subsequent cryotoming, followed 

by micro- spectroscopic mapping of the cryosections to get spatial information on growth 

heterogeneity within the colonies [1]. 

We also used confocal Raman micro-spectroscopy(cR-MSP) as a method to investigate 

heterogeneity in microbial cell populations at the single cell level. cR-MSP is an evolving technique 

for the rapid, non-invasive chemical imaging of composite microscopic structures in biological 

materials at a sub-micron spatial resolution, which allows the investigation of both, the spatial 

distribution of cell components, and their chemical or structural nature. We used the same test 

organisms as for the IR-MSP experiments and detected pronounced intra- and inter-cellular 

heterogeneity in macro-colonies and even liquid cultures cultivated under laboratory conditions. 

Our Raman imaging results unequivocally proved the presence of heterogeneous multicomponent 

systems in genetically homogeneous microbiological samples [2]. Thus, cR-MSP 

qualifies as an ideal tool to identify and visualize single component distribution within multicomponent 

systems without the need of any dye or contrasting agents. 

References 

[1] N.A. Ngo Thi, D. Naumann, Anal. Bioanal. Chem. 387, 1769-1777 (2007). 

[2] A. Hermelink, A. Brauer, P. Lasch, D. Naumann, Analyst 134, 1149-1153 (2009). 

26


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Application of raman spectroscopy to study the interaction of 

anti-cancer drugs with cells 

HAQ NAWAZ 1 , AIDAN MEADE 1 , FRANCK BONNIER 1 , 

FIONA LYNG 2 AND HUGH J. BYRNE 1 

1. Focas Institute, Dublin Institute of Technology; 

2. Radiation and Environmental Science Centre (RESC), Focas Institute, Dublin Institute of Technology, 

Dublin 8, Ireland. 

Early assessment of chemotherapeutic drug efficacy is very important for the better development 

and screening of new drug candidates. A cost-effective way for determining drug efficacy at the 

early stage of drug development is to understand its mechanism of action at the cellular and 

molecular level. Vibrational spectroscopy, particularly Raman spectroscopy, has demonstrated 

potential in the analysis of chemical changes resulting from external agents at the sub-cellular 

level. In the current studies, the interaction of cis-Diamminedichloroplatinum (II) (Cisplatin) with 

the human lung adenocarcinoma, A549, cell line was investigated using Raman spectroscopy. The 

identification of molecular changes occurring in the cells after interaction with the drug was made 

on the basis of spectral changes. The results obtained were also compared with the classical 

cytotoxicological test, the MTT assay. Raman spectroscopic analysis of isolated DNA, RNA and 

protein from cisplatin treated A549 cells will be carried out. This study will be extended to 

anticancer drugs of varying mechanism of action and different cell lines. 

27


PB 

Differentiation of cells from peripheral blood by 

vibrational spectroscopic imaging 

U. NEUGEBAUER 1 , J. H. CLEMENT 2 , T. BOCKLITZ 3 , C. KRAFFT 1 AND J. POPP 1,3 

1. Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena, 

Germany 

2. Department of Internal Medicine II, Friedrich-Schiller-University Jena, 

Erlanger Allee 101, D-07740 Jena, Germany 

3. Institute of Physical Chemistry, Friedrich-Schiller-University Jena, 

Helmholtzweg 4, D-07743 Jena, Germany 

Cancer can originate in almost any part of the body. Once a tumour is formed, it can invade the 

surrounding tissue and disseminate cells via the blood and lymph vessels all over the body. It is of 

high diagnostic interest to separate and differentiate the tumour cells from the healthy cells in 

peripheral blood [1]. However, established cell sorting techniques are not yet sufficient to fulfil this 

demand. Raman and IR spectroscopic imaging offer high potential for non-destructive and label 

free characterization of cells [2-4]. Here, we apply those techniques to identify different cells that 

can be found in peripheral blood: tumour cells, fibroblasts and leukocytes. Breast carcinoma 

derived tumour cells (MCF-7, BT-474) were grown in cell culture as well as lymphoblasts (OCI). 

Leukocytes were isolated from healthy donors. All cells are prepared on CaF2 slides for 

measurement. Differentiation of these cells can be achieved using a combination of multivariate 

tools including k-means cluster analysis, hierarchical cluster analysis and principal component 

analysis. With the help of supervised statistical methods it is possible to identify the cells with 

more than 90% accuracy based on their vibrational spectra. 

References 

Fig. 1: Averaged Raman spectra of different cells whose white light 

images and Raman maps (amid I band) are shown on the right 

[1] J. H. Clement, M. Schwalbe, N. Buske, K. Wagner, M. Schnabelrauch, P. Görnert, K. O. Kliche, K. Pachmann, W. 

Weitschies, K. Höffken, J Cancer Res Clin Oncol 132, 287-292 (2006). 

[2] P. Rösch, M. Harz, K. D. Peschke, O. Ronneberger, H. Burkhardt, J Popp Biopolymers 82, 312-316 (2006). 

[3] C. Krafft, R.Salzer, S. Seitz, C. Ern, M. Schieker Analyst 132, 647-653 (2007). 

[4] C. Krafft, T. Knetschke, R. Funk, R. Salzer Analytical Chemistry 78, 4424-4429 (2006). 

28


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Cooling of E. coli Tryptophanase leads to a covalent bond 

scission and cold lability 

A. KOGAN 1 , GA. Y. GDALEVSKY 1 , R. COHEN-LURIA 1 , Y. 

GOLDGUR 1 , R. S. PHILLIPS 2 , O. ALMOG 3 , A. H. PAROLA 1 

1 Department of Chemistry, Ben-Gurion University of the Negev, POB 653, Beer-Sheva 84105, Israel. 

2 Departments of Chemistry and Biochemistry and Molecular Biology, University of Georgia, Athens, GA 

30602, USA. 

3 Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, 

Israel. 

Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme 

is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent 

tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 

at the active site. The incubation of holo E. coli Trpases at 2ºC for 20 h results in breaking this enamine 

bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This 

sequence of events is termed cold lability and its understanding bears relevance to protein stability 

and shelf life [1, 2]. We studied the reversible cold lability of E. coli Trpase and its Y74F, C298S and 

W330F mutants. In contrast to the holo E. coli Trpase all apo forms of Trpase dissociated into dimers 

already at 25ºC and even further upon cooling to 2ºC. The crystal structures of the two mutants, Y74F 

and C298S in their apo form were determined at 1.9Å resolution. These apo mutants were found in an 

open conformation compared to the closed conformation found for P. vulgaris in its holo form. This 

conformational change is further supported by a high pressure study. We suggest that cold lability of 

E. coli Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants 

is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight 

assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic 

interactions which facilitate PLP release. The hydrophobic interactions around the central tetrameric 

axis overshadow the effect of point mutations and may account for the differences in the dissociation 

of E. coli Trpase to dimers and P. vulgaris Trpase to monomers. 

References 

[1] N. Tsesin, A. Kogen, G. Y. Gdalevsky, J.-P. Himanen, R. Cohen-Luria, A. H. Parola, Y. Goldgur, and O. Almog, Acta 

Cryst. Sec. D63, 969–974 (2007). 

[2] O. Almog, A. Kogen, M. de Leeuw, G. Gadlevsky R. Cohen-Luria, and A. H. Parola. Biopolymers, 89(5), 354-359, 

(2008). 

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Diagnosis of prostate cancer by Raman spectroscopy 

correlated to tissue biomarkers 

K. W. C. POON 1 , O. HOWE 2 , F. LYNG 2 AND H. J. BYRNE 1 

1. FOCAS Institute, Dublin Institute of Technology, Camden Row, Dublin 8, Dublin, Republic of Ireland 

2. Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Camden 

Row, Dublin 8, Dublin, Republic of Ireland 

Annually, over 670,000 men are diagnosed with prostate cancer worldwide, accounting for 

approximately one quarter of all diagnosed cancer cases in men[1]. The advent of the PSA 

(Prostate Specific Antigen) blood test has revolutionised early detection of prostate cancer, 

however, it is unable to distinguish between benign growths and aggressive cancerous forms. 

Definitive diagnosis is only possible through a biopsy of the prostate, from which a skilled 

histopathologist is required to observe changes related to tissue/cell ultrastructure based on the 

universally accepted Gleason tumour grading system. This system focuses on a variety of complex 

transformations in the prostate glandular morphology known to occur during the progression of 

the cancer. However, Gleason scoring suffers from intra/inter observer subjectivity, with studies 

showing 70.8% of prostate cancer biopsies being misgraded [2]. Raman spectroscopy has been 

shown to discriminate between normal and pathological tissue samples initially processed by 

formalin fixation and paraffin embedding (FFPE)[3]. As tissue samples sourced from hospitals and 

medical institutions use FFPE as the standard processing technique, it is important to correlate 

spectroscopic studies with such protocols. Results will be presented regarding the capability of 

Raman spectroscopy to distinguish between FFPE tissue sections of normal, benign prostate 

hyperplasia (BPH) and prostate cancer in combination with multivariate analysis. The results will 

also be correlated with various immunohistochemical stains such as AMACR (α-methylacyl-CoA 

racemase) and their relationship to existing data regarding aberrant G2 radiosensitivities in 

patients.[4] 

References 

[1] Cancer Research UK CancerStats Key Facts: Prostate Cancer. 

http://info.cancerresearchuk.org/cancerstats/types/prostate/ (25/05/2009). 

[2] Lattouf, J.-B.; Saad, F., Gleason, BJU International, 90, 694-698 (2002). 

[3] Lyng, F. M.; Faoláin, E. Ó.; Conroy, J., et al., Experimental and Molecular Pathology, 82, 121-129, (2007). 

[4] Howe, O.; O'Malley, K.; Lavin, M., et al., Radiation Research, 164, 627-634, (2005). 

30


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Resistance to irradiation of micro-algae growing in 

the storage pools of a nuclear reactor investigated by 

NMR and neutron spectroscopies 

C. RIVASSEAU 1 , E. FARHI 2 , M. GROMOVA 1 , J. OLLIVIER 2 , R. BLIGNY 1 

1. Laboratoire de Physiologie Cellulaire Végétale, CEA, CNRS, 17 rue des Martyrs, 38054 

Grenoble cedex 9, France 

2. Institut Laue Langevin, BP156, 38042 Grenoble cedex 9, France 

Life develops everywhere on earth including extreme environments. The extremophile species 

include for instance organisms surviving high salt concentrations, toxic metals, high pressures, 

acidity, low or high temperatures [1,2], vacuum, and desiccation. Additionally, some bacteria, 

among which the champion Deinococcus radiodurans, have been found to exhibit an 

extraordinary ability to survive ionizing irradiation, up to 15 kGy. We discovered an ionizingirradiation 

tolerant micro alga growing in the light water cooling systems and in the nuclear waste 

fuel elements storage pool of the ILL research reactor. This unicellular green micro alga belonging 

to the Chlorophyceae class lives just a few meters away from the core in a highly radio-active 

environment and in a very poor nutrient medium. It is the only eukaryotic autotrophic organism 

to grow in such environment, for at least 15 years, although contamination possibilities from 

outside environment are not unusual. This adaptation indicates an irradiation tolerance capacity 

higher than that of other eukaryotes, and a probable adaptation to this environment. Moreover, 

after being exposed to up to 20 kGy, these algae re-colonize their culture medium within some 

weeks [3]. The ability of this micro algae whose genus has been identified to tolerate ionizing 

irradiation has never been reported. More generally, there is little knowledge on algae resistance to 

gamma irradiation. The study of this micro-alga is consequently of peculiar interest, all the more as 

it concerns a eukaryotic organism which is much more complex and fragile than a bacteria. 

Understanding mechanisms involved in resistance to irradiation in this organism might bring new 

insights into radiation response of other eukaryotic organisms including animals. We investigated 

the effects of irradiation on the micro algae physiology, metabolism, and internal dynamics. Micro 

algae were stressed with irradiation doses up to 20 kGy (2 Mrad), their survival was assessed as a 

function of the irradiation dose, and changes in metabolism, internal dynamics, and structure 

brought by irradiation were studied respectively using nuclear magnetic resonance (1H NMR) and 

neutron backscattering on the IN13 instrument at the ILL (elastic incoherent backscattering as a 

function of temperature) [3]. It was found that this Chlorophyceae green micro alga resisted high 

γ-irradiation doses, up to 6 kGy. Higher doses induced a sensible decrease in the survival rate, but 

the micro algae colonies may still recover after a month as shown by the recovery of the 

physiological parameter photosynthesis. Even though the associated tolerance mechanism is not 

known today, we performed spectroscopic investigations which provided a few macroscopic 

indicators about the cell behaviour during irradiation. […] 

References 

[1] Tehei M, Franzetti B, Wood K, Gabel F, Fabiani E, Jasnin M, Zamponi M, Oesterhelt D, Zaccai G, Ginzburg M, 

Ginzburg B-Z, Proc. Natl Acad. Sci. 104 (2007) 766 

[2] Tehei M, Zaccai G, Biochimica et Biophysica Acta 1724 (2005) 404 

[3] Farhi E, Rivasseau C, Gromova M, Compagnon E, Marzloff V, Ollivier J, Boisson AM, Bligny R, Natali F, Russo D, 

and Couté A, J. Phys.: Condens. Matter 20 (2008) 104216 

31


PB 

Reagent-free quantitative determination of ingredients 

of body and related fluids in real time using ATR-FTIR 

spectroscopy 

A. ROTH 1 , O. KLEIN 1 , I. KLEIN 2 AND W. MÄNTELE 1 

1. Institute of Biophysics, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 1, 60438 Frankfurt am 

Main, Germany 

2. Innovectis GmbH, Altenhöferallee 3, 60438 Frankfurt am Main, Germany 

Especially for realtime point-of-care diagnostics of fluids in the medical environment there is a 

demand for portable analysis solutions without any sample preparation or reagents. The technique 

of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy offers these 

possibilities for various ingredients of body fluids such as whole blood, blood plasm or urine and 

related fluids such as blood filtrates. By using multivariate mathematical models like partial leastsquares 

regression one can determine simultaneously the absolute concentration of relevant 

components, such as for example albumin, cholesterol, glucose, hemoglobin, total protein, 

triglycerides and urea in whole blood or creatinine, phosphate, urea and uric acid in urine [1]. The 

absolute precision and reproducibility of the determined values is thereby only limited by the 

precision of the reference analysis used for calibration and complies to clinical standards. Based on 

a compact, portable FT-IR spectrometer two special ATR measuring cells were developed, one for 

small discrete sample volumes of less than 10 µL to be used for a single blood droplet drawn from 

the fingertip. A second cell was developed for quasi-continuous flow-through measurements in a 

continuous flow at flow rates up to 1000 mL/min in intense care to analyse time-dependent 

concentration changes. Time profiles are possible because one measurement only takes about one 

minute. Both measuring cells will be evaluated in the clinical routine in comparison to 

conventionally used laboratory equipment working with reagents and requiring some sample 

preparation. Furthermore, the system can easily be adapted to other types of fluids simply by 

recalibration. For example, a flow-through cell was recently used to determine the main 

parameters of beer [2]. 

References 

[1] G. Hosafci, O. Klein, G. Oremek, W. Mäntele, Anal Bioanal Chem 387, 1815–1822 (2007) 

[2] O. Klein, W. Mäntele, GIT Labor-Fachzeitschrift 10, 824-827 (2005) 

32


PB 

Breast tissue diagnosis by Raman spectroscopy 

J. SURMACKI 1 , B. BROśEK-PŁUSKA 1 , J. JABŁOŃSKA 2 , R. KORDEK 2 AND H. ABRAMCZYK 1,3 

1. Technical University of Lodz, Chemistry Department, Laboratory of 

Laser Molecular Spectroscopy, 93-590 Lodz Wroblewskiego 15 str 

Poland, e-mail: jsurmacki@mitr.p.lodz.pl 

2. Department of Oncology, Medical University of Lodz, 93-509 Lodz 

Paderewskiego 4, Poland 

3. Max-Born-Institute Fax: +49 30 6392 1409 Max-Born-Str. 2A 12489 

Berlin Germany, e-mail: abramczy@mbi-berlin.de 

We present Raman spectra on ex-vivo samples from breast tissue (for 134 patients) and compare 

them with the results of histopathological analysis to estimate the sensitivity and specificity of the 

method. The results demonstrate the ability of Raman spectroscopy to accurately characterize 

breast cancer tissue and distinguish between normal, malignant and benign types. We provide 

evidence that carotenoids and lipids of the tissue play an essential role as a Raman biomarkers. 

The normal tissue has characteristic bands: C-C (1156cm -1) and C=C (1520cm -1) stretching bands of 

carotenoids and at 2840-2900 cm -1 for C-H symmetric and asymmetric bands of lipids (fat) which 

are not observed in malignat tumor tissue. 

Fig. 1 Raman spectra and histopatological image: a) normal tissue, 

b) carcinoma ductale G3 infiltrans mammae. 

The support from MEXC-CT-2006-042630 and Nr3 T11E 04729 projects is acknowledged. 

References 

[1] H. Abramczyk, I. Placek, B. BroŜek-Płuska, K. Kurczewski, Z. Morawiec, M. Tazbir, J. Mol. Liquid 141, 145-148 

(2008) 

[2] H. Abramczyk, I. Placek, B. BroŜek-Płuska, K. Kurczewski, Z. Morawiec, M. Tazbir, Spectroscopy 22, 113-121 

(2008) 

[3] H. Abramczyk, I. Placek, B. BroŜek-Płuska, K. Kurczewski, Z. Morawiec, M. Tazbir, ISRAPS Bulletin, 20, no 1 

(2008) 

[4] H. Abramczyk, J. Surmacki, B. BroŜek-Płuska, Z. Morawiec, M. Tazbir, J. Mol. Struc. 924-926, 175-182 (2009) 

33


PB 

Affinity towards sulphation of wool and silk fibres 

P. TADDEI 1 , M. TSUKADA 2 , T. ARAI 2 AND G. FREDDI 3 

1. Biochemistry Department, University of Bologna, Via Belmeloro 8/2, 

Bologna, I-40126, Italy 

2. Division of Applied Biology, Faculty of Textile Science and 

Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386-8567, 

Japan 

3. Stazione Sperimentale per la Seta, Via G. Colombo 83, Milan, I-20133, 

Italy 

Wool keratin and silk fibroin are excellent biopolymers with outstanding properties that make 

them extremely valuable in biomedical field. Coatings are commonly applied to the surface of 

materials to improve their surface properties. The biocompatibility and non-immunogenicity of 

silk proteins should allow their application as coatings for biomedical implants, potentially as 

anticoagulants, and either promoters or inhibitors of cell adhesion. Incorporation of sulphate and 

sulphonate groups confers anticoagulant and anti-thrombogenic properties to polymers. In view of 

widening the biomedical utility of natural polymers as biomaterials, here we present a 

comparative vibrational study on wool, B. mori and A. pernyi silk fibroin fibres sulphated with 

chlorosulphonic acid in pyridine, which is known to enhance the yield of sulphation. Our aim is to 

prepare sulphated fibres by keeping the intrinsic fibre properties and texture unchanged, using 

short reaction times (i.e. 3h). The fibres were analyzed by Attenuated Total Reflectance, ATR/FT- 

IR and FT-Raman spectroscopy to comparatively elucidate the affinity for sulphate groups, the 

mechanism and the mode of linkage, the amino acid side-chains involved, and the possible 

conformational changes caused by sulphation. Among the analysed samples, the vibrational 

spectra of sulphated wool fibres showed the most pronounced changes, suggesting the highest 

affinity towards sulphation. New bands in the 1300-1200 and 1100-900 cm -1 ranges were assigned 

to the formation of alkyl and aryl sulphate salts, sulphonamides, sulphoamines and covalent arylalkyl 

sulphates. Vibrational spectra revealed the occurrence of a certain fibre degradation as well 

as rearrangements with consequent changes in secondary structure, conformation of disulphide 

bridges and tyrosine environment. The amino acid residues mainly involved in sulphation were 

identified as serine, threonine, tyrosine and tryptophan. Upon sulphation, the IR and Raman 

spectra of B. mori silk fibroin fibres showed analogous changes although less pronounced than for 

wool fibres. No significant changes were detected for A. pernyi silk fibroin fibres: only slight 

conformational rearrangements were observed. The reactivity towards sulphation was found to 

decrease along the series: wool > B. mori silk fibroin > A. pernyi silk fibroin, in agreement with the 

weight gain measurements which decreased along the same series. These results can be explained 

in relation to the different composition of the analysed fibres. Wool fibres were characterised by 

the highest content of the potentially reactive sites; evidently, these groups had also a good 

accessibility for the sulphating agent. 

34


PB 

Zn-metallothionein complexes studied by Raman 

spectroscopy 

A. TINTI1 , A. TORREGGIANI, 2 J. DOMÈNECH, 2, 3 

1. Dept. Biochemistry “G. Moruzzi”, University of Bologna, Via Belmeloro 8/2 , Bologna, I-40126, Italy 

2. ISOF-CNR, Via P. Gobetti 101, Bologna, I-40129, Italy 

3. Dep. Genètica, Univ. Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain 

Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins, with an exceptional heavy 

metal coordination capacity. These metal chelating peptides play an active role in zinc homeostasis. 

Although their high heterogeneity, structural and functional studies have been mainly devoted to 

vertebrate and fungal MTs. Participation of metal ligands other than Cys and the presence of 

secondary structure elements in metal-MT complexes are fairly unknown, especially in nonvertebrate 

MTs. Six in vivo-synthesized Zn-MTs, representative of different MT families (mollusc, 

insect, nematode, echinoderm, plant and vertebrate) were studied by analytic and spectroscopic 

techniques. The examined MTs contain from 43 to 73 amino acids, among which at least a 30% are 

Cys, whereas very few are aromatic residues. Zn(II)-MT complexes were heterologously 

synthesized in E.coli, to obtain aggregates representative of those formed in biological systems. 

These complexes contain variable amounts of metal and sulfide ions, quantitatively evaluated by 

analytical measurements. The formation of more than one species (S 2- -containing and S 2- -devoid 

complexes), revealed by ESI-MS spectra, is a constant for all these MTs. The analysis of the 

Raman spectra gave information about the secondary structure of the metal-MT aggregates. For all 

the MT examined, a relevant contribution of β-turns was shown, whereas in all cases the α-helix 

content resulted almost negligible. As regards Cys sulfurs, almost all Cys residues present in MTs 

resulted to be involved in the metal coordination, as indicated by the presence of several bands 

attributable to the metal-S stretching modes at low wavenumbers (< 500 cm -1 ). The high number of 

νM-S bands and their broadening suggest the formation of different metal centres. Significantly, 

Raman bands useful as markers of sulfide bridging ligands were identified. In Zn-CeMT2 (from 

C.Elegans) and Zn-QsMT (from plant – Quercus suber) the eventual participation of the His residue 

in metal binding was evaluated through a curve fitting analysis of the 1630-1565 cm -1 Raman 

spectral range. His residues resulted to be mainly coordinated in Zn-CeMT2 (≈ 90%), whereas in 

Zn-QsMT His is mainly present as free tautomer (≈ 90%) [1]. Raman spectroscopy showed to be a 

powerful technique, able to provide information on the state of the Cys sulfur (metal coordinated 

and oxidised), the metal binding environment (i.e. the participation of His in metal coordination), as 

well as on the secondary structure. 

References 

[1] A. Torreggiani, J. Domenech, S. Atrian, M. Capdevila, A. Tinti, Biopolymers 89 (2008) 1114-1124. 

35


PB 

Raman-based diagnostic tools to distinguish anemias and 

to evaluate the expression ratio of S and C hemoglobins 

ALESSANDRO VERGARA 1 , VISHNU PULLY 2 , GIULIA RUSCIANO 3 , ANNA BALSAMO 1 , 

FILOMENA SICA 1 , ANTONELLO MERLINO 1 , MICHELA GROSSO 4 , ANTONIO SASSO 3 , 

LELIO MAZZARELLA 1 , CEES OTTO 2 

1 “Paolo Corradini” Dept. of Chemistry, University of Naples Federico II. anna.balsamo@unina.it. 

2 MESA+ Institute, Biophysical Engeenering Group, University of Twente, Enschede, Netherland. 

3 Dept. of Physical Sciences, University of Naples Federico II. 

4 Dept. of Biochemistry and Medical Biotechnologies, University of Naples Federico II. 

Cellular imaging techniques based on vibrational spectroscopy (Raman) are powerful tools in cell 

biology because the molecular composition of sub-cellular compartments can be visualized 

without the need for labeling [1]. Moreover resonance Raman (RR) spectroscopy is an ideal 

method to elucidate the structural characteristics of heme domains in hemoproteins, directly 

monitoring the iron-axial ligand bond strength. The information provided is important in 

understanding the mechanisms of protein control of heme reactivity [2]. In this context, Optical 

Tweezers (OT) have revealed a formidable tool to select and manipulate single particles, included 

cells or cellular organelles, allowing a rapid identification of the changes, at single cell level, in 

response to external agents, environmental stress, or in presence of cellular disorders [3]. 

Haemoglobinopathies are inherited disorders defined as qualitative or quantitative deficiency of 

haemoglobin (Hb) in red blood cells (RBCs). Herein we analyze anemic RBCs with two human Hb 

variants, HbS and HbC (Gluβ6Val and GlubβLys, respectively). A comparative study in a family 

(mother, father and daughter, respectively heterozygous for HbS, HbC and Hb C+S) shows that 

different Raman images and mechanical properties of healthy and anemic RBCs could help to 

evaluate the expression ratios between HbS and HbC, and correlate these data with varied clinical 

conditions. 

References 

[1] van Manen H.J., et al., Proc. Natl. Acad. Sci. 2005, 102, 10159. 

[2] Vitagliano L., et al., J. Am. Chem. Soc. 2008, 130, 10527. 

[3] De Luca A.C., et al., Opt. Exp. 2008, 16, 7943. 

AV and LM acknowledge PRIN 2007 funds. 

36


PB 

Study of controlled release of model dyes and drugs 

from poly(D,L lactic-co-glycolic) acid nanoparticles and 

rod-shaped PLGA pellets by fluorimetry, analytical 

ultracentrifugation and fluorimetry imaging 

V.VOGEL, 1 K. LANGER, 2 W. MÄNTELE 1 

1. Institute of Biophysics, Johann Wolfgang Goethe-University, D-60438 , 

Frankfurt , Germany 

2. Institute of Pharmaceutical Technology and Biopharmacy, WWU Münster, 

Correnstrasse 1, 48149 Münster Germany 

The development of sustained release systems capable of delivering low molecular drug and 

peptides over extended periods of time is an actual problem for a variety of biomedical 

applications. Poly(D,L lactic-co-glycolic acid) nanoparticles (PLGA-NP) and rod-shaped PLGA 

pellets represent promising biodegradable carriers for controlled drug delivery. We present the 

analysis of model dye-loaded PLGA nanoparticles and rod-shaped PLGA pellets as models for a 

drug delivery system. Model drug loaded PLGA nanoparticles were prepared by a solvent 

extraction/evaporation method with polyvinyl alcohol (PVA) as an emulsifier and rod-shaped 

PLGA pellets were prepared by compression. Loaded PLGA nanoparticles and rod-shaped PLGA 

pellets were characterized for drug encapsulation efficiency and in vitro drug-release kinetics by 

fluorimetry, analytical ultracentrifugation and fluorimetry imaging. Carbocyanine fluorescence 

dyes and model drugs were used for release kinetic measurements. 

References 

[1] M.Holzer, V.Vogel, W.Mäntele, D Schwartz, W.Haase, K.Langer, Eur J Pharm Biopharm., 2009, 72, 428 – 437. 

37


PB 

Extended X-ray Absorption Fine Structure Studies of 

Formation of Iron/GT56-252 Complex 

DONGHUI WANG, EUGENE ZHOROV, AHARON COHEN 

Analytical R&D, Genzyme Corporation 

211 Second Ave, Waltham MA 02451, USA 

Binding chemistry between Iron (III) and a carboxylic acid (GT56-252) in aqueous and bovine 

serum was studied by using XAFS (X-Ray Absorption Fine Structure) at the National Synchrotron 

Light Source, Brookhaven National Laboratory in Upton, NY, USA. Significant changes of Iron 

coordination chemistry upon addition of GT56-252 were experimentally confirmed, and such 

changes can be well explained by the formation of Iron/GT56-252 complex with both Fe-O and Fe- 

N bonds. In addition, a basic environment (pH = 9) and a higher temperature (up to 60°C) 

appeared to be favorable for the complex formation. 

The following work will be presented to understand the binding between Iron and GT56-252: 

1. Iron coordination in aqueous solution before and after GT56-252 addition. 

2. Iron coordination in bovine serum before and after GT56-252 addition. 

3. pH effect on Fe/GT56-252 formation in aqueous solution. 

4. Temperature effect on Fe/GT56-252 formation in aqueous solution. 

5. Reaction rate of iron with GT56-252 in bovine serum. 

The major findings from this study are: 

1. Iron/GT56-252 complex was formed in either aqueous solution or in bovine serum. 

2. In iron (III) aqueous solution, upon addition of GT56-252, a significant change of Iron 

absorption edge position was observed. This confirmed the formation of Iron/GT56-252 complex. 

Same Iron/GT56-252 complex was formed in bovine serum. 

3. The Iron/GT56-252 complex formation appears to increase with increasing pH. 

4. Increasing temperature appears to be favorable for the complex formation. 

5. In bovine serum, equilibrium in the complex formation was achieved in the first few 

minutes if not faster 

38

Dielectric spectroscopy 13 th ECSBM −−−− Book of Abstracts 

PB 

Thermally Stimulated Depolarization Currents (TSDC): 

a Sensitive Technique for Analyzing Proteins Structure 

M. G. BRIDELLI, R. CAPELLETTI, E. POLVERINI, AND R. BEDOTTI 

Department of Physics, University of Parma, Via G. P Usberti 7/A, Parco 

Area delle Scienze, Parma I-43100, Italy 

Thermally Stimulated Depolarization Current (TSDC) technique has been widely employed to 

monitor the state of water in proteins and other biomolecules at low hydration levels [1]. The 

technique, thanks to its very high sensitivity, is able to detect the solvent organization around the 

“solute” structure by distinguishing water dipoles in terms of their order degree and mobility, 

although the relation between the biopolymer conformation and the structure of water network 

has not yet been completely understood. As concerns proteins, the typical TSDC spectrum (a plot 

of the depolarization current as a function of the temperature) consists, as a rule, of two main 

peaks, generally called Low Temperature (LT) and High Temperature (HT) peak, respectively, the 

former being related to water molecules located in the grooves of the folded structure and the 

latter to the external ones, decorating the polypeptide surface. However, the differences in the 

position, amplitude, and shape of the two peaks for different proteins are meaningful (see Figure) 

and have suggested a novel application of the technique in the biophysical field: the H2O dipoles, 

belonging to the solvation shell, are exploited as probes to gain information about the structural 

and conformational features of proteins. On these bases, correlation between TSDC peak 

parameters and polypeptide structure has been recently proven for Lysozyme in native and 

amyloid form [2]. The present work can be regarded as a survey of the TSDC spectra recorded for 

a number of proteins with different structures and conformations : the contributions, deriving both 

from the different secondary structure and from the “preference” of water molecules to be bound 

at particular sites, are qualitatively distinguished. Complementary FTIR and CD spectra are 

measured and the results are compared to those already published. 

Fig. 1 – TSDC spectra of β-lactoglobulin ( ), pepsin ( ) and α-chymotrypsin (----) pellets prepared at 

relative humidity h=0.92. Polarization range (displayed by segment and arrow ): T p=300K, T f = 85K. 

References 

current / pA 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

50 100 150 200 250 300 350 

Temperature /K 

[1] M.G. Bridelli, R. Capelletti, F. Maraia, C.Mora and L.Pirola, J. Phys. D: Appl. Phys. 35, 1039-1048 (2002). 

[2] M. G. Bridelli and R. Capelletti, Spectroscopy 22, 165-176 (2008). 

39

Dielectric spectroscopy 13 th ECSBM −−−− Book of Abstracts 

PB 

Modulation Instability of Patterns in a passive nonlinear 

photorefractive ring oscillator 

M. K. MAURYA*, T. K. YADAV AND R. A. YADAV 

Lasers and Spectroscopy Laboratory, Department of physics 

Banaras Hindu University, Varanasi-221005, India 

*Email: mahendrabhu@gmail.com 

The study of Modulation instabilities in nonlinear optical systems has been a very active field of 

research in recent years [1-2]. These instabilities arise from diffraction phenomena affecting the 

light beam as it propagates through the nonlinear medium and lead to the appearance of 

nontrivial e.g., non-Gaussian structures of the electromagnetic field in a plane orthogonal to the 

direction of propagation [3]. From the application point of view, such system can be engineered to 

perform as useful devices for switching, storing and manipulating information [3-5]. We have been 

studied theoretically, the modulation instability and it has been also studied the process of 

modulation instability in a temporally incoherent cavity below the cavity threshold and also the 

influence of various parameters such as cavity length, intensity feedback and spatial frequency on 

the gain factor and spectral density have also been studied. 

References 

[1] T. Carmon, H. Buljan, and M. Segev, Opt. Express 12, 3481-3487 (2004). 

[2] Hrvoje Buljan, Marin Soljacic, Tal Carmon, and Mordechai Segev, Phys.Rev. E 68, 016616 (2003). 

[3] F.T. Arecchi, S. Boccaletti and PL. Ramazza, Phy.Rep. 318, 1 (1999) 

[4]T. Carmon, M. Soljacic, and M. SegevPhy. Rev. Lett. 89, 183902 (2002). 

[5]L. A. Lugiato and R. Lefever, Phy.Rev. Lett. 58 ,2209-2211 (1987). 

40

DNA & RNA 13 th ECSBM −−−− Book of Abstracts 

PB 

1H NMR studies on the binding of pixantrone 

anticancer drug to d(ACGATCGT)2 

N. ADNAN 1 , D.P.BUCK 2 AND J. G. COLLINS 2 

1. Dept. of Physical and Mathematical Sciences, University of New South 

Wales @ ADFA, Via Northcott Drive, Canberra, 2600, Australia. 

The study of binding of Pixantrone to a range of oligonucleotide d(ACGATCGT)2 has been studies 

by 1H NMR spectroscopy. The non-exchangeable base and sugar protons of the d(ACGATCGT)2 

and drug/DNA sample were assigned by the combined use of one and two dimensional NMR 

spectroscopy[1][2].The addition of the anticancer drug pixantrone to d(ACGATCGT)2 induced 

significant changes in chemical shifts of base and sugar resonances of the resonances from A1, T8, 

T5, G7, G3, C2 residues from oligonucleotide.In NOESY spectra of oligonucleotide bound with 

either pixatrone, NOEs were observed from C2H5, C6H5 protons of the nucleotides residues. This 

result indicate that the pixantrone bind at or near the C.G in the major groove. This result suggests 

that the pixantrone selectively bind at C.G in the major grooves of DNA. A model was built for the 

binding of pixantrone to d(ACGATCGT)2; drug was inserted manually into the free 

oligonucleotide, pixantrone intercalates between the base pairs of G7, G6 and G3, C2 due to absence 

of NOEs between these pairs.The result presented here suggests that pixantrone may be excellent 

diagnostic agents for the d(ACGATCGT)2. 

Fig. 1 

Residue H8/H6 H2 H1' H2' H2'' Methyl 

A1 8.10(-0.12) 8.03(-0.14) 6.08 (-0.15) 2.53(-0.1) 2.69(-0.08) 

C2 7.34(-0.06) 5.28(-0.09) 2.16(-0.1) 2.32(-0.03) 

G3 7.84(-0.06) 5.47(-0.22) 2.62(-0.09) 2.62(-0.09) 

A4 8.26(0.04) 7.65(-0.17) 6.19(-0.1) 2.60(-0.05) 2.60(-0.16) 

T5 7.06(-0.1) 5.89(-0.04) 1.95(-0.08) 2.39(-0.04) 1.35 

C6 7.40(-0.02) 5.73(0) 1.90(-0.05) 2.39(-0.11) 

G7 7.90(-0.07) 5.94(-0.13) 2.60(-0.08) 2.70(-0.05) 1.64 

T8 7.30(-0.11) 6.16(-0.09) 2.24 (-0.03) 2.24(-0.02) 1.46 

1H DNA Assignments of the DNA resonances at R=2.3 molar ratio pixantrone-controls DNA- 

NOESY (35⁰C) 

References 

[1] J.G. Collins, Biochemistry. Int., Vol.16. 819-828 (1988). 

[2] Belinda S. Parker, Trevor Buley, Ben J. Evison, Suzanne M.Cutts, Greg M.Neumann, Magdy N. Iskander and Don 

R. Phillips, A molecular understanding of Mitoxantrone-DNA adducts formation, Department of Biochemistry, La 

Trobe University, Victoria (2004). 

41


PB 

Virus + Metalloprotein Electrostatic Assembly 

P. NUNES 1 , C.SANTOS 1 , P. EATON 2 AND C. J. CALDEIRA 1 

1. REQUIMTE FCTUNL Monte de Caparica 2825-516 Caparica, Portugal 

2. REQUIMTE FCUP Campo Alegre 4169-007 Porto, Portugal 

This work aims to develop and study novel magnetically induced and self-assembly materials. The 

application of external field (electric, mechanic or magnetic) imposed a macroscopic direction to 

the autonomous organized ensemble of molecules. We developed a partially aligned material 

based on the electrostatic assembly of Pf1 virus and a metalloprotein (cytochrome c). Pf1 virus can 

be magnetically aligned ( > 7 Tesla) in order to produce an aligned matrix. These bacteriophage 

has a tubular structure (l=2μm, Ø=6,7 nm) with a single strain DNA inside and a capsid of 7620 

subunits of 46 amino acids (one subunit per DNA Base). The virus surface is strongly negatively 

charged (-0,475e/nm 2, pI=4.0), due to the presence of 3 aspartic acid and no other charged residues 

per subunit. Cytochrome c (horse heart) is a simple protein (12384 Da) with one heme and positive 

surface charge (pI = 10.2). Experimentally we have determined a reversible binding between Pf1 

bacteriophage and cytochrome c at ~120mM of ammonium acetate pH 6,8 at 25ºC with a specific 

stochiometry producing a neutrally charged complex. This complex was characterized by Atomic 

Force Microscopy (Figure 1), Dynamic Light Scattering, Zeta Potential, Diffusion-Ordered 

Nuclear Magnetic Spectroscopy, and thermodynamic statistical Monte Carlo simulations. This 

work is supported by POCTI/QUI/58973/2004 FCT – MCTES. 

Fig. 1 Assembly of Pf1 bactriophage and cytochrome c observed in a liquid cell AFM. 

References 

[1] L. Makowski, D.L. Caspar and D.A. Marvin,. J. Mol. Biol. 140,149–181. (1980). 

[2] Pil J. Yoo, Ki Tae Nam,Angela M. Belcher, and Paula T. Hammond, Nanoletters, 8 1081-1089, (2008). 

[3] Nam, K. T., Kim, D. W., Yoo, P. J., Chiang, C. Y., Meethong, N., Hammond, P. T., Chang, Y. M., Belcher, A. M. 

Science, 312, 885-888, (2006). 

[4] Yoo, P.J., Nam, K., Qi, J., Lee, S.K., Park, J., Belcher, A.M., Hammond, P.T., Nature Materials,5, 234-240. (2006). 

[5] J. Caldeira, J. L. Figueirinhas, C. Santos and M. H. Godinho J. Mag. Resonan., 170, 213-219, (2004). 

[6] Marie Adams, Zvonimir Dogic, Sarah L. Keller , Seth Fraden. Nature, 393, 349-352, (1998). 

42


PB 

SEIRA markers of DNA condensations by La 3+ ions. 

G. DOVBESHKO1 , O. FESENKO 1, O. GNATIUK1, B. DRIBINSKY 2 AND N. KASYANENKO 2 

1. Institute of physics of National Academy of Science of Ukraine, 46 

Prospect Nauki, Kiev 03028, Ukraine. 

2. Dept. of Physics, Saint-Petersburg State University, 7-9 

Universitetskaya nab., Saint-Petersburg 199034, Russia. 

Spectroscopic features of DNA condense by La 3+ from aqueous solution was studied by SEIRA 

(Surface enhanced infrared absorption) spectroscopy and AFM methods. Concentration of La 

changes from 2*10 -6 M to 5*10 -5 M. The conformation states of DNA during the processes of 

condensation could be characterized as B-form. The position of antisymmetrical phosphate band in 

condense DNA equals to 1224-1229 cm -1. We have registered the increase intensity about two times 

for both vibration of phosphates and bases, as well as increase of shoulder of band responsible of 

base paring in the region of 1710-1700 cm -1. A number of water molecules per nucleotide decreases 

in condense state of DNA for all La concentration about two times and maximum of stretching 

vibration of NH-OH band shifts in high frequency region from 3360 to 3400 cm -1. Under the 

smallest concentration of La the Z – form of sugar-phosphate backbone is registered and then 

under more La concentration it converted in B-form. La improves an ordering in double helix 

structure. 

Acknowledgments. The authors are grateful for the support to this research by Ukraine 

program “Nanostructured Systems, Nanomaterials, Nanotechnology”. 

References 

Absorption 

2,0 

1,8 

1,6 

1,4 

1,2 

1,0 

0,8 

0,6 

0,4 

0,2 

0,0 

1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 

Wavenumber, cm -1 

43 

La 2*10-6 

La 8*10-6 

La 2*10-5 

La 5*10-5 

контроль 

Fig. 1 – SEIRA spectra of DNA in the region 1800-800 cm -1 . 

Spectra are normalized according to band at 3400 cm -1 . 

[1] G. I. Dovbeshko, O. P. Gnatyuk, V. I. Chegel, Y. M. Shirshov, D. V. Kosenkov, E. A. Andreev, H. A. Tajmir-Riahi, P. 

Litvin, Semiconductor physics, quantum electronics & optoelectronics 7 (3), 318-325 (2004). 

[2] V.A. Bloomfield, Current Opinion in Structural Biology, 6, 334–341 (1996).


PB 

Pyrene-modified RNA – conformation and dynamics 

U. FOERSTER 1 , C. GRUENEWALD 2 , J.W. ENGELS 2 AND J. WACHTVEITL 1 

1. Institute of Biophysics, Goethe-University, Frankfurt, Max-von-Laue- 

Str. 1 , 60438 Frankfurt, Germany 

2.Institute of Organic Chemistry and Chemical Biology, Goethe- 

University, Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt, Germany 

We present the photophysics of pyrene-modified RNA examined by several ultrafast spectroscopic 

techniques. Attached to the 2-position of the adenine base, pyrene has proven to be a useful 

marker to monitor the structure and dynamics of RNA. Singly and doubly modified strands as 

well as the pyrene modified adenine have been studied via time resolved absorption and 

fluorescence spectroscopy. The measurements showed a complex photophysical behavior of the 

label, indicative for a structured excited state leading to several decay. The pyrene-adenine 

photophysics is solvent dependent and influenced by the sequence of the adjacent nucleobases[1]. 

Doubly modified RNA was prepared in a way, that a pyrene was attached to each strand of a 

duplex, allowing the two dyes to approach each other closely enough for excimer interaction upon 

hybridization[2]. A detailed study of the melting process of singly and doubly modified RNA 

duplexes revealed details about the unfolding dynamics of the RNA strands as well as structural 

information concerning the pyrene label. Time-resolved studies of the doubly modified RNA 

showed a coexistence of monomer and exciplex states of the pyrene label. 

References 

Fig. 1 Left: Fluorescence upconversion of the pyrene-modified base 

in isopropanol at two different excitation wavelengths. The data 

show an excitation energy dependent decay from a higher excited 

state into the fluorescent state. Right: Melting curve of doubly 

pyrene-modified RNA. Evaluation of fluorescence wavelength 

corresponds to a loss of the pyrene-pyrene interaction, which 

happens earlier than the melting of the whole RNA strand, that 

causes a change in fluorescence intensity. 

[1] U. Foerster, C. Gruenewald, J.W. Engels, J. Wachtveitl, submitted (2009) 

[2] C. Gruenewald, T. Kwon, N. Piton, U. Foerster, J. Wachtveitl, J.W. Engels, Bioorg. Med. Chem 16, 19-26 (2008). 

44


PB 

FRET and pulsed EPR spectroscopies to investigate 

biochemical and structural features of DNA lesions 

D. GASPARUTTO 1 , M. FLAENDER 1 , S. GAMBARELLI 2 AND G. SICOLI 2 

1. Lab. Lésions Acides Nucléiques and 

2. Lab. Résonances Magnétiques, SCIB – UMR E3 CEA-UJF-CNRS / INAC / CEA Grenoble, 17 avenue des 

Martyrs 38054 Grenoble, France 

DNA is the target of several endogenous and exogenous processes that give rise to a large set of DNA 

lesions that include base modifications, base losses, cross-links and strand-breaks. Such genetic alterations if 

not repaired are able to evolve in lethal and mutagenic events during DNA replication and transcription 

steps. To determine the toxicity and the reparability of the different DNA lesions, we have developed new 

tools consisting in DNA fragments that contain a selective lesion at a defined site together with fluorescent 

or nitroxide dyes. Such double-stranded modified DNA probes have been used in FRET- or pulsed EPRbased 

spectroscopic studies to evaluate the structural modifications and the reparability of the targeted DNA 

lesions. The FRET-based probes containing a lesion were labelled with a fluorophore and a quencher (Fig. 

part (A)) and then used as substrates for DNA repair enzymes. The fragments resulting from the cleavage at 

the lesion site are rapidly separated from each other because of their low melting temperatures. 

Consequently, the excision of the lesion from the DNA leads to an irreversible fluorescent enhancement. This 

constitutes the basis for a quantitative analysis of DNA repair activities [1, 2]. Regarding the pulsed EPR 

approach, double electron-electron resonance (DEER) was applied to determine nanometre spin-spin 

distances on DNA duplexes that contain selected structural alterations (Fig., part(B)). The lesion-induced 

conformational changes observed are consistent with previous results obtained by NMR and 

crystallographic methods, and prove the capability for pulsed EPR to probe structural modifications within 

damaged DNA [3]. Finally, the results obtained with such DNA biosensors coupled with advanced 

spectroscopy methods are powerful to assess the biological significance of DNA alterations and then better 

understand their potential implications in mutagenic and carcinogenic processes. 

(A) 

Strand cleavage 

by repair enzyme 

(at the damaged site) 

Fig. 1 Principle of the FRET-based DNA repair assay (A) and the pulsed EPR approach (B) 

developed to evaluate biological and structural features of DNA lesions. 

References 

Fluorescence 

quenching or not 

(On/Off probe) 

[1] A. Chollat-Namy et al., Chemistry of Nucleic Acid Components- Coll. Symp. Series (2005) 

[2] D. Jary et al., Nanotech. (2006) 

[3] G. Sicoli et al., Nucleic Acids Research (2009) 

45 

(B) 

Damaged site 

Interspin distance 

measurements 

by EPR


PB 

Circular dichroism and conformational 

polymorphism of DNA 

I. KEJNOVSKÁ, M. VORLÍČKOVÁ, D. RENČIUK AND J. KYPR 

Institute of Biophysics, v. v. i. Academy of Sciences of the Czech Republic, Královopolská 135, CZ- 

612 65, Brno, Czech Republic 

Circular dichroism (CD) spectroscopy is a very effective method for DNA conformational studies 

[1,2]. Conformational properties of DNA include the B-family of structures, A-form, Z-form, 

guanine quadruplexes, cytosine quadruplexes, triplexes and other less characterized structures [3- 

5]. CD spectroscopy is extremely sensitive and relatively inexpensive. This fast and simple method 

can be used at low as well as high DNA concentrations and with short as well as long DNA 

molecules. The samples can easily be titrated with various agents to cause conformational 

isomerizations of DNA. The course of detected CD spectral changes makes possible to distinguish 

between gradual changes within a single DNA conformation and cooperative isomerizations 

between discrete structural states. It enables measuring kinetics of the appearance of particular 

conformers and determination of their thermodynamic parameters. In careful hands, CD 

spectroscopy is a valuable tool for mapping conformational properties of particular DNA 

molecules. Due to its numerous advantages, CD spectroscopy significantly participated in all basic 

conformational findings on DNA. 

Acknowledgements: 

The study was granted by Grant Agency of the Czech Republic (grant 204/07/0057 and 204/00/D012), and Grant 

Agency of the Academy of Sciences of the Czech Republic (grant IAA 500040903 and IAA 100040701). 

References 

[1] J. Kypr, I. Kejnovská, D. Renčiuk, M. Vorlíčková, Nucleic Acids Res. 37, 1713-1725 (2009). 

[2] M Vorlíčková, J. Kypr, V. Sklenář, “Nucleic Acids: Spectroscopic methods”, in Encyclopedia of Analytical Science, 

Second Edition edited by P.J. Worsfold, A. Townshend and C.F. Poole, Oxford: Elsevier, 391 (2005). 

[3] I. Kejnovská, M. Tůmová, M. Vorlíčková, Biochim. Biophys. Acta 1527, 73-80 (2001). 

[4] M. Vorlíčková, J. Chládková, I. Kejnovská, M. Fialová, J. Kypr, Nucleic Acids Res. 33, 5851-5860 (2005). 

[5] D. Renčiuk, M. Zemánek, I. Kejnovská, M. Vorlíčková, Biochimie 91, 416-422 (2009). 

46


PB 

Complexation DNA with an anti-cancer drug 

M. KLAJNER 1, 2 , C. GAIDDON 3 , C. SIRLIN 4 , P. HEBRAUD 1 AND S. HARLEPP 1 

1. I.P.C.M.S.,UMR7504, Université de Strasbourg, Strasbourg, France 

2. Institute of Physics, Wroclaw University of Technology, Wroclaw, Poland 

3. INSERM U692-Université de Strasbourg, Signalisations Moleculaires et 

Neurodegenerescence, Strasbourg, France. 

4. Unite Mixte de Recherche 7513-Université de Strasbourg, Laboratoire de Syntheses Metallo- 

Induites, Strasbourg, France. 

Organometallic compounds based on Platinum are used as anticancer drugs. Nevertheless, they 

exhibit strong second side effects, and affect the neuronal activity. Other new organometallic 

derivatives, and among them those based upon Ruthenium are good candidates as alternative 

anti-cancer drugs. In our presentation, we study one of these Ruthenium Derived Compounds (1-4) 

(RDCs), and show that it exhibits a strong affinity to DNA. Using Förster Resonant Energy 

Transfer (FRET), and mechanical stretching of a single DNA molecule, we show that the 

complexation of DNA with RDC is reversible, measure the affinity constant of RDC for DNA and 

we lastly probe the conformational changes on DNA structure induced by the adsorption of RDC. 

References 

[1] C. Gaiddon, P. Jeannequin, P. Bischo, M. Pfeffer, C. Sirlin, J. Loeffler, Journal of Pharmacology and Experimental 

Therapeutics 315, 1403 (2005). 

[2] L. Leyva, C. Sirlin, L. Rubio, C. Franco, R. Le Lagadec, J. Spencer, P. Bischo, C. Gaiddon, J.-P. Loeffler, M. Pfeffer, 

<strong>European</strong> Journal of Inorganic Chemistry, 3055-3066 (2007). 

[3] W. H. Ang, E. Daldini, C. Scolaro, R. Scopelliti, L. Juillerat-Jeanneret, P. J. Dyson, Inorganic chemistry 45, 9006- 

9013 (2006). 

[4] R. E. Morris, R. E. Aird, Pdel S. Murdoch, H. Chen, J. Cummings, N. D. Hughes, S. Parsons, A. Parkin, G. Boyd, D. 

I. Jodrell, P. J. Sadler, Journal of Medical Chemistry 44, 3616–3621 (2001). 

[5] J. Weiss, FASEB Journal 11, 835 (1997). 

[6] J. Genereux, K. Augustyn, M. Davis, F. Shao, J. Barton, Journal of American Chemical Society 130, 15150 (2001). 

47


PB 

Cisplatin to DNA binding kinetics monitored by 

Raman spectroscopy 

V. MASEK 1 , P. MOJZES 2 , J. PALACKY 2 AND P. ANZENBACHER 1 

1. Dept. of Pharmacology, Faculty of Medicine and Dentistry, Palacky 

University, Hnevotinska 3, Olomouc CZ-77515, Czech Republic 



It is widely accepted that the DNA is the pharmacological target for anticancer drug cisplatin [1] 

and that the cytotoxicity of cisplatin and other platinum compounds is primarily determined by 

their DNA adducts [2]. Cisplatin forms covalent bonds preferentially with nucleophilic sites on 

guanine residues present in all DNA. As cisplatin is a bifunctional agent, it is able to bind to one or 

two sites in a DNA chain. Structures, other physical properties of these adducts and their 

recognition by the components of 

downstream cellular systems processing 

DNA damage have been intensively 

studied [4-6]. In this study we employed 

Raman spectroscopy to monitor kinetics 

of cisplatin binding to model DNA (800bp 

long fragments of polynucleotide poly 

(dG-dC)*poly (dG-dC) and Escherichia coli 

plasmid pUC19, 2686 bp). The set of data 

was analysed using Factor Analysis 

(Singular Value Decomposition) and the 

results show that the process of cisplatin 

to DNA binding cannot be described only 

by two subspectra. It means that Raman 

spectroscopy can distinguish phases of 

formation of monofunctional and 

bifunctional adducts. 

Acknowledgement 

This work was supported by the Academy of Sciences of the Czech Republic (KAN 200200651), the Ministry of 

Education of the Czech Republic (MSM6198959216 and MSM0021620835), the Grant Agency of the Charles University 

in Prague (204/2006/B-FYZ/MFF) and the Grant Agency of the Czech Republic (203/07/0717). 

References 

[1] N. P. Johnson, J.-L. Butour, G. Villani, F. L. Wimmer, M. Defais, V. Pierson, and V. Brabec, Metal antitumor 

compounds: The mechanism of action of platinum complexes, Prog. Clin. Biochem. Med. 10 (1989) 1-24. 

[2] V. Brabec, DNA modifications by antitumor platinum and ruthenium compounds: their recognition and repair, Prog. 

Nucleic Acid Res. Mol. Biol. 71 (2002) 1-68. 

[4] S. M. Cohen, and S. J. Lippard, Cisplatin: From DNA damage to cancer chemotherapy, Prog. Nucleic Acid Res. Mol. 

Biol. 67 (2001) 93-130. 

[5] M. A. Fuertes, C. Alonso, and J. M. Perez, Biochemical modulation of cisplatin mechanisms of action: Enhancement 

of antitumor activity and circumvention of drug resistance, Chem. Rev. 103 (2003) 645-662. 

[6] D. Wang, and S. J. Lippard, Cellular processing of platinum anticancer drugs, Nature Reviews Drug Discovery 4 

(2005) 307-320. 

48


PB 

The influence of nitric oxide on DNA glycosylase-MutY 

S.MĘCZYŃSKA-WIELGOSZ, J. SADŁO AND M. KRUSZEWSKI 

Institute of Nuclear Chemistry and Technology, 16 Dorodna Str, 

Warsaw, 03-195 Poland 

The nitric oxide (NO ●) is a very reactive molecule produced in biological systems from the arginine 

by synthases of the nitric oxide. The nitric oxide and his derivatives can directly affect 

macromolecules appearing in cells , eg. on proteins and DNA. The nitric oxide can also influence 

indirectly on homeostasis of the cell, bearing on the expression of genes, the activity of proteins or 

upsetting the ionic balance. One of postulated mechanisms of the influence of the nitric oxide on 

proteins is freeing of ions of iron from iron-sulphur clusters, which are sensitive to the activity of 

free radicals. Proteins containing iron universally appear in living organisms. An example of such 

proteins are DNA glycosylases, enzymes participating in the repair DNA with the excision of the 

base and preventing genotoxic and mutagenic results of the formation of oxidized bases. Some 

glycosylases, eg. endonuclease III and the MutY protein and their mammiferous homologues, 

contains iron-sulphur cluster [4Fe-4S]. MutY protein, one of the enzymes, so called „the GO 

system”, preventing to disadvantageous results of the formation 8-oxoguanine. As contrasted with 

other DNA glycosylases, the MutY protein removes mismatched, but undamaged adenine from 8oxoguanine:adenine 

mispairs. The MutY protein contains [4Fe-4S] iron-sulphur cluster 

coordinated by cysteines. The cluster confers a secondary structure of the enzyme and is probably 

responsible for the regulation of its activity. Recent research showed that the nitric oxide and its 

oxidized derivatives, i.e.peroxynitrite, can knock out the iron-sulphur cluster from proteins or to 

form dinitrosyl iron complexes (DNIC) containg such clusters. This modification causes 

conformational changes of the protein and likely changes or inhibits its biological activity. In 

presented work we examined the influence of the nitric oxide on the conformation and the activity 

of the MutY protein. We explored the interactions of the nitric oxide and its derivatives with ironsulphur 

cluster in vitro and in vivo by means of EPR, UV/VIS spectroscopy and its influence on 

biological activity of MutY. We showed that treating of E.coli producing MutY, DEANO (nitric 

oxide donor) causes the formation of the dinitrosyl iron complexes which disappear during the 

isolation of the protein from these bacteria. Native MutY protein has a typical UV-Vis absorption 

spectrum of an oxidized [4Fe-4S] cluster with a major absorption peak at 419 nm The absorption 

peak at 419 nm has been attributed to the thiolate-to-iron charge transfer transitions of the [4Fe-4S] 

clusters. Treating of the MutY protein with different concentrations of the nitric oxide did not 

cause the disappearance the absorption peak characteristic for the iron-sulphur cluster. We 

compared also the biological activity of native protein and isolated from bacteria treated nitric 

oxide donor. Nitric oxide treatment (50 micromoles) diminish the activity of native MutY protein 

in vitro , however the activity of the protein isolated from bacteria treated 50 micromolar nitric 

oxide remains unchanged. 

49


PB 

Structure of a 25mer HCV RNA studied by Raman 

spectroscopy 

P. CARMONA 1 , M. MOLINA 2 AND A. RODRIGUEZ-CASADO 3 

1. Instituto de Estructura de la Materia (CSIC), Serrano 121, 28006 Madrid, Spain. 

2. Departamento de Química Orgánica I, Escuela Universitaria de Óptica, 28037 Madrid, Spain. 

3. IMDEA Alimentación, 28049 Madrid, Spain 

Hepatitis C virus (HCV) is an RNA virus that causes acute and chronic liver disease in humans, 

including chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1]. The HCV genome is a 

single-stranded RNA molecule of positive polarity which contains a highly conserved 5’ 

untranslated region (5’UTR) of 341 nucleotides in length. We carried out here the Raman analysis 

of an HCV RNA 25mer (1-25 nucleotides) comprising the domain I of the 5’UTR, in aqueous and 

heavy water solution. The intensity ratio (R) 1.25 obtained of lines near 810 cm -1 (diester OPO 

stretching vibration) and 1100 cm -1 (PO2 - symmetric stretching vibration) in Raman spectra is an 

indication that around 76% of the bases in this HCV RNA are ordered (with the C3’-endo/anti 

ribofuranose pucker), and A-form secondary structure is found to be predominant. The Raman 

data show that some uridine and guanosine nucleoside can adopt C2’-endo/anti conformations, 

which are characteristic of the backbone B-form, and C3’-endo/syn conformations respectively. This 

can be attributable to positions where chain foldings switch abruptly from helical to looped. The 

analysis of the D2O solution Raman spectrum through curve fitting, in the C=O region, to a sum of 

Gaussians representing paired and unpaired structures indicate that 6 out of 9 guanine residues 

are Watson-Crick base paired. The spectral data suggest that this HCV RNA 25mer sequence 

adopts a hairpin form whose secondary structure is consistent with that proposed on the basis of 

phylogenetic comparisons whit other viral RNAs [2]. 

References 

Raman Intensity/Arbitr. Units 

1705 

1599 

1575 

1530 

1482 

1600 1400 1200 1000 800 600 

Wavenumber/cm -1 

Fig. 1 – Raman spectrum of the 25mer HCV RNA in H 2O buffer. 

[1] F. Penin, J. Dubuisson, F.A. Rey D. Moradpour, J.M. Pawlotsky. Hepatology, 39, 5-19 (2004) 

[2] M. Kunkel, M. Lorinczi, R. Rijnbrand, S.M. Lwmon, S.J. Watowich, J. Virol, 75, 2119-2129 (2001). 

1368 

1416 

1317 

1250 

50 

1098 

1043 

983 

921 

867 

812 

783 

724 

668 

629


PB 

Dimeric structure of quadruplex-forming thrombinbinding 

aptamer in complexes with cationic porphyrin 

J. PALACKÝ AND P. MOJZEŠ 

Institute of Physics, Faculty of Mathematics and Physics, Charles 


Guanine quadruplexes are structurally interesting inter- or intramolecular four-stranded DNA 

structures with biological and technological importance [1]. Depending on the oligonucleotide 

sequence and physico-chemical conditions, wide range of G-qaudruplexes with different 

stoichiometry, topology and structural features can be formed. Despite numerous studies, certain 

ambiguities and discrepancies still exist even about structures of the most frequently studied 

quadruplexes, notably if determined by various experimental methods at diametrically different 

conditions. For instance, the thrombin-binding aptamer 5’-d(GGTTGGTGTGGTTGG)-3’ (TBA), 

generally taken as a prototype of a chair-type intramolecular quadruplex [1], was recently 

suggested to exist as a bimolecular complex in aqueous solutions [2]. Present work reports certain 

hints in favor of dimerization or bimolecular nature of TBA quadruplexes in aqueous solutions, as 

deduced from structural features of the TBA complexes with tetracationic Cu 2+-5,10,15,20-tetrakis- 

[4-(N-methylpyridyl)]-porphine (CuTMPyP4). Structure and thermal stability of the TBA 

oligonucleotide and of its complexes with CuTMPyP4 were thoroughly studied under various 

conditions (concentration, CuTMPyP4/TBA ratio, ionic strength) by a combination of UV-vis 

absorption, CD and Raman spectroscopies in the presence of three cations (K +, Na +, Li +) differing 

in ability to induce G-quadruplex. It was found that CuTMPyP4 bound to TBA in the presence of 1 

– 100 mM K + displays large conservative CD profile within its Soret band (~430 nm), the feature 

characteristic of porphyrin-porphyrin interactions on a chiral matrix reported previously for 

CuTMPyP4 complexes with single-stranded poly(dA) [3]. The same effect of considerably lower 

extent was observed in the presence of 100 mM Na + but not with Li +. To relate consistently selfassociation 

of the CuTMPyP4 molecules bound to TBA-quadruplexes with the 1:1 complex 

stoichiometry reported previously [4], formation of a specific binding site accommodating two 

porphyrins has been suggested due to dimerisation of two intramolecular quadruplexes or 

formation of an antiparallel bimolecular quadruplex. Melting profiles based on the CD spectra 

revealed apparently lower thermal stability of the porphyrin conservative CD features 

(Tm~39.6±0.8°C) than of the quadruplex ones (Tm~47.5±0.8°C). In the presence of 1 - 100 mM K +, 

binding of the CuTMPyP4 has no effect on the quadruplex thermal stability. On the contrary, 

binding to the Na +-induced quadruplex destabilizes its structure and thermal stability, the effect 

being much greater at low Na + concentration. In the presence of 10 – 100 mM Li +, no spectral signs 

of quadruplex persisting after addition of the CuTMPyP4 were found, the spectral features of the 

complex being consistent with partial intercalation of the porphyrin into the single-stranded TBA. 

References 

[1] Quadruplex Nucleic Acids; S. Neidle, S. Balasubramanian (Eds.), RSC Publishing, 2006. 

[2] M. Fialova, J. Kypr, M. Vorlickova, Biochem. Biophys. Res. Commun. 344, 50-54 (2006). 

[3] R. F. Pasternack, R. A. Brigandini, M. J. Abrams, A. P. Williams, E. J. Gibbs, Inorg. Chem. 29, 4483-4486 (1990). 

[4] M. Toro, R. Gargallo, R. Eritja, J. Jaumont, Anal. Biochem. 379, 8-15 (2008). 

51


PB 

Spectroscopic Characterization of Biologically Active 

Complex of Mn(II) with 5-Bromouracil 

HITESH KUMAR 1 , MEENAKSHI SHARMA 1 , B.K. RAI 2 , J.K. VATS 1 , T.V.S. ARUNMURTHHY 3 , M. 

ALCOLEA PALAFOX 4 , AND V.K. RASTOGI 1 

1. Department of Physics, CCS University, Meerut-250 004, India 

2. Department of Chemistry, L.N.T. College, Muzaffarpur-842 001 (Bihar), India 

3. Department of Physics, SSGM College of Engineering, Shegaon (MS), India 

4. Departamento de Química-Física I, Facultad de Ciencias Químicas, Universidad 

Complutense, Madrid-28040, Spain, alcolea@quim.ucm.es 

The structure of molecules is primary basis for understanding and predicting their physical, 

chemical, biological and material properties [1]. The essential biological importance of uracil and 

its derivatives has motivated a number of recent studies on the structure and spectroscopy of these 

molecules [2, 3]. DNA contains uncommon nucleotides usually in very small amount. 5bromouracil 

is one of the well known uncommon nucleotide base have the ability to bind metal or 

to bind to tissues via metals. As the metal complexes play an important role in the biological 

activity of drugs, therefore in the present study, we report the isolation and characterization of the 

complex of Mn(II) with 5-bromouracil. The metal complex was isolated by mixing the hydrated 

manganese nitrate solution (1 ml mole) prepared in ethanol (14 ml)-water (100 ml) mixture and hot 

alcoholic solution of 5-bromouracil (1 ml mole). The resulting mixture was heated to about 65 °C 

and the NaOH was added to adjust the pH to about 4-6. The white precipitate then obtained was 

filtered, washed several times with ethyl alcohol from the same solvent. The residue was dried in 

an oven at about 90 °C. The complex formed possesses 1:1 stoichiometry and the chemical 

composition corresponds to molecular formula [Mn(L)(OH)2.3H2O]; L = C4H2N2O2Br. On 

comparing the spectra (IR, Raman) of 5-BrU with the spectra of its Mn(II) complex it was found 

that the spectra of the complex show some bands shifts relative to free 5-BrU in the C=O region. 

The intensity of some bands also changes. The ν(C4=O) mode, which appears at 1655 cm —1 in 

spectra of 5-BrU, shifted to lower frequency by ~ 40 cm —1, suggesting that 5-BrU coordinates to 

Mn(II) through C4=O group as the bands due to ν(C2=O), ν(N-H) and ν(C5-Br) modes remain 

almost unaltered. The appearance of a bond ~ 350 cm —1 due to ν(M-O)aquo confirms the presence of 

coordinated water molecules in the complexes. On the basis of above studies an octahedral 

polyatomic structure have been suggested for the complex. The polymerization involves bridging 

through OH. 

References 

[1] P. Pulay, X. Zhou, G. Fogarasi. Recent Experimental and Computational Advance in Molecular Spectroscopy, p-99, 

(Kluwer Academic Publishers Netherlands) (1993). 

[2] M.A. Palafox, G. Tardajos, A. Guerrero-Martinez, V.K. Rastogi, D. Mishra, S.P. Ojha, W. Kiefer, Chem. Phys., 340, 

1227 (2007). 

[3] V.K. Rastogi, M.A. Palafox, L. Mittal, N. Peica, W. Kiefer, K. Lang, S.P. Ojha, J. Raman Spectroc., 38, 1227 (2007). 

52


PB 

Study of influence of NIR radiation on herring sperm DNA 

by ATR-FTIR, UV spectroscopies and DSC 

K. SZYMBORSKA-MALEK 1 , B. CZARNIK–MATUSEWICZ 2 AND M. KOMOROWSKA 3 

1. Institute of Physical and Theoretical Chemistry, Wroclaw University 

of Technology, Wybrzeze Wyspianskiego 27, Wroclaw, Poland 

2. Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 

Wroclaw, Poland 

3. Institute of Biomedical Engineering and Instrumentation, Wroclaw 

University of Technology, Wybrzeze Wyspianskiego 27, Wroclaw, 

Poland 

Our previous studies let us suppose that the Near Infrared (NIR) radiation of aqueous solution of 

biomolecules causes specific modification of water structure in their vicinity. This hypothesis was 

already evidenced both for whole cells (erythrocytes and liposomes) and single molecules 

(phenylalanine) [1],[2]. It was postulated that the NIR modified water enhances protonation of 

investigated molecules that facilitates their aggregation and conformational changes [3]. The 

experimental phenomena were confirmed by the results of quantum chemical calculations[4]. 

Presented results concern investigation of the influence of the NIR radiation, for different time of 

exposition, on thermal stability of herring sperm DNA. Studied samples were irradiated for 

periods of 5, 10, and 20 minutes. UV (ultraviolet) and ATR-FTIR (attenuated total reflectance- 

Fourier transform infrared) measurements were performed in the temperature range from 25 oC to 

90 oC to monitor the conformational changes in this biomolecule. Also DSC (differential scanning 

calorimetry) data were collected for the unirradiated and irradiated samples of DNA. The 

presented study has provided a new insight into following points: 

1. UV melting profiles show three stages of DNA denaturation at T1 (25 – 50 oC), T2 (50 - 70 oC) and 

T3 (70 - 95 oC), however only T2 is sensitive to NIR radiation. Results obtained from DSC 

measurements are consistent with conclusions gained from the UV experiment. 

2. PCA (principal components analysis) of ATR-FTIR spectra measured in function of 

temperature and irradiation time also points the three stages of the melting process. Effect of 

short time of exposition to NIR differs markedly from control, 10, and 20 minutes irradiated 

samples. 

3. ATR-FTIR spectra allow us to conclude that the water activity in the base, phosphate and sugar 

region depends on NIR radiation period. Short time irradiation (5 min.) modifies mainly 

hydration of the base and phosphate groups what protects DNA against denaturation. Longer 

time of exposition to NIR influences dehydrated bases, phosphates and sugar groups what 

promotes the process of denaturation. 

References 

[1] M. Komorowska, A. Cuissot, A. Czarnoleski, W. Bialas, J. Photochem. Photobiol. B, 6S, 93-100 (2002) 

[2] S. Olsztynska, M. Komorowska, N. Dupuy, Appl. Spectrosc., 60(6), 648-652 (2006) 

[3] S. Olsztynska, N. Dupuy, L. Vrielynck, M. Komorowska, Appl. Spectrosc., 60(9), 1040-1053 (2006) 

[4] S. Olsztynska, K. Szymborska, M. Komorowska, J. Lipinski, Acta Bioeng. Biomech., 10, 45-49 (2008) 

53


Where Structural Alterations Start for the Allosteric Mechanism 

of Catabolite Repressor Protein (Crp); from a Monomeric Apo- 

Crp Structure or from a Dimer Apo-Crp Structure? 

PB 

B.A. FAS 1, T. HALILOGLU 1, Y. TUTAR 2 

1. Dept. of Chemical Engineering, Bogazici University, Bebek, Ýstanbul, 34342 

2. Dept. of Chemistry, Cumhuriyet University, Merkez, Sivas, 58140, TURKEY 

The adaptability of cells to its environment is essential for viability. Therefore, transcription 

initiation mechanism of lac operon in E. coli under short glucose supply has been investigated for 

over three decades [1, 2]. Here, 80 ns molecular dynamics (MD) simulations are performed for 

each of the two modeled Catabolite Repressor Protein (CRP) structures: I) monomeric CRP and ii) 

dimeric CRP to search for the apo conformation of CRP. The simulations suggest that the 

monomeric state is a plausible structure for the apo state in comparison with the experimental 

observations of salt bridge formation and the secondary structure content of the apo form. Also, 

the hinge movement towards the C-proximal domain is observed in the simulations agree with the 

experimental findings. Nevertheless, the simulation also suggest that the dimerization stabilizes 

the domains and the association of the two domains through the DNA binding site. All these with 

the biochemical data point out that the allosteric mechanism of transcription initiation may rely on 

monomer-dimer equilibrium. cAMP binding shifts the equilibrium to right and increases specific 

DNA binding affinity. On the other hand, protein-DNA complex dissociate by stepwise removal of 

the monomers upon an increase at local DNA concentration. Key residues involved in this 

mechanism are Glu72,Arg82, and Arg123 [3]. First two residues form a salt bridge and this process 

expels Arg123 side chain to solvent at apo state, which is only observed during the monomer 

simulation. Upon cAMP binding Glu72 forms a salt bridge with Arg123 and Arg82 forms an 

interior pair with the phosphate oxygen of the cAMP. In the absence of cAMP and DNA, Glu72- 

Arg123 salt bridge is observed in the dimer simulation. Previous mechanisms can not explain 

milimolar cAMP concentration during activation process which is quite different than in vivo 

cAMP concentration however our data provides a plausible model for allostery with in the limits 

of micromolar ligand concentration [4, unpublished data]. 

References 

[1] Y. Tutar, Cell Biochem Func 26, 399-405 (2008). 

[2] S. R. Tomlinson, Y. Tutar, J. G. Harman, Biochemistry, 45, 13438-13446 (2006) 

[3] Y. Tutar, J. G. Harman, Arch Biochem Biophys 15, 217-223 (2006). 

[4] Y. Tutar, Protein J, 27, 21-29, 212 (2007). 

54

Nanotechnology 13 th ECSBM −−−− Book of Abstracts 

PB 

Simulation of dissipative dynamics in light-harvesting 

complexes: eliminating fitting parameters from 

Redfield equations 

A. S. BELOV, V.V. EREMIN 

Chemistry Dept., Moscow State University, Leninskiye Gory 1/3, 

Moscow, 119991, Russia 

The equations of dissipative dynamics, given by Redfield theory [1], are widely used for modeling 

the excitation energy or charge transfer in light-harvesting complexes (LHCs), including natural 

photosystems. Since Redfield theory is a “system plus reservoir” model, its practical application 

heavily depends on the availability of the data on the so-called spectral density – the generalized 

properties of the reservoir. Being an elusive property, the spectral density requires nontrivial 

techniques for its determination [2]. We propose an improvement of the commonly used model, 

which allows to overcome this difficulty. We begin with microscopic description of the reservoir, 

which is considered as a homogeneous isotropic media, filled with harmonically coupled point 

charges. After assuming the dipole-dipole approximation for all interactions and averaging with 

respect to reservoir coordinates, we arrive at the analytical expression for the elements of the 

Redfield tensor [3]. This expression can be linked to the vibrational spectrum of the reservoir, 

which makes it possible to express dynamic rate constants in terms of electronic and photophysical 

properties of the chromophores within the light-harvesting complex. The accuracy of the proposed 

model was checked by comparing calculated rates with those obtained from femtosecond 

experiments for the LH2 antenna of purple bacteria [4-6]. The model was recognized to be valid for 

semi-quantitative simulations. The sources of simulation inaccuracies and prospects of further 

development are discussed. 

References 

[1] U. Weiss, “Quantum Dissipative Dynamics 2 nd edition”, Singapore: World Scientific Publishing Co., 9 (1999) 

[2] V. I. Novoderezhkin, M. A. Palacios, H. van Amerongen, and R. van Grondelle J. Phys. Chem. B 108 10363-10375 

(2004) 

[3] Belov A.S., Eremin V.V. Moscow Univ. Bullet. Chemistry series in press (2009) 

[4] J. M. Salverda, F. van Mourik, G. van der Zwan, and R. van Grondelle J. Phys. Chem. B 104 11395-11408 (2000) 

[5] Y.-Z. Ma, R. J. Cogdell, T. Gillbro J. Phys. Chem. B 101 1087-1095 (1997) 

[6] H.-M. Wu, S. Savikhin, N. R. S. Reddy, R. Jankowiak, R. J. Cogdell, W. S. Struve, and G. J. Small J. Phys. Chem. 

100 12022-12033 (1996) 

55


Development and validation of an improved Oxygen Radical 

Absorbance Capacity assay using biomimetic systems 

PB 

C. M. CASTRO, M. LÚCIO, M. A. SEGUNDO, S. REIS AND J. L. F. C. LIMA 

REQUIMTE, Serviço de Química Física, Faculdade de Farmácia da 

Universidade do Porto, Rua Aníbal Cunha 164, 4099-033 Porto, Portugal 

In the past years, the importance of antioxidants in the protection of organisms or tissues has 

become evident. This statement is supported by studies performed in a variety of areas, including 

physiology, pharmacology, nutrition and even food processing [1]. In all these areas of research 

fast, reliable methods for antioxidant assessment are needed [2]. Generally, the ideal method for 

determination of antioxidant properties should assess the effect of a compound/sample in reaction 

conditions that mimic those found when oxidative stress is induced in vivo by reactive nitrogen 

species (RNS) and reactive oxygen species (ROS). At the present moment there is no standard 

methodology to fulfill this task [3], but several assays have been proposed to take this role, 

including the ORAC (Oxygen Radical Absorbance Capacity) assay [4]. This assay is based on the 

intensity of fluorescence decrease of the target/probe along time under reproducible and constant 

flux of peroxyl radicals, generated from the thermal decomposition of 2,2′-azobis(2amidinopropane) 

dihydrochloride (AAPH) in aqueous buffer. In the presence of a sample that 

contains chain-breaking antioxidants, the decay of fluorescence is inhibited. However, this method 

has been criticized as the fluorescent probe (fluorescein) does not mimic any target structure found 

in vivo. Hence, in order to address this problem and make conditions more closer to those found in 

vivo, biomimetic systems, such as liposomes have been employed [5]. In this case, liposome works 

as a model of biological membranes, containing lipids that are also a target for peroxidation from 

ROO·. In the present work a novel competitive liposome method is compared to the conventional 

ORAC method. Assays were performed in a microplate reader and involved the addition of AAPH 

solution to reaction media containing fluorescein and antioxidant compound (at different 

concentrations). Multilamellar vesicles prepared from phosphatidylcholine were also added in the 

competitive assay. Preliminary results using Trolox, a water soluble analogue of vitamin E, shown 

that the reaction time, defined as the time necessary to attain a value of fluorescence intensity 

lower than 1% of the initial value, was significantly higher for the competitive assay when 

compared to the conventional approach. This is due to the lower concentration of ROO· available 

to interact with the antioxidant compound, as part of ROO· is consumed by their attack to the 

lipids present in the liposome. This was confirmed when analysis time values similar to those 

verified in the conventional assay were attained when the AAPH concentration was increased 3 

times. Biologically relevant compounds with known antioxidant activity (reduced glutathione, uric 

acid, ascorbic acid) are presently under study by application of both methodologies, with 

evaluation of different parameters (area under curve, lag time, slope of fluorescein decay, endpoint 

time) derived from the fluorescence intensity profile obtained along reaction time. 

References 

[1] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 4 th ed., Oxford: Oxford University Press, 

(2007). 

[2] L.M. Magalhães, M.A. Segundo, S. Reis, J.L.F.C. Lima, Anal. Chim. Acta 613, 1-19 (2008). 

[3] R.L. Prior, X.L. Wu, K. Schaich, J. Agric. Food Chem. 53, 4290-4302 (2005). 

[4] A.N. Glazer, Methods Enzymol. 186, 161-168 (1990). 

[5] M. Lúcio, H. Ferreira, J.L.F.C. Lima, S. Reis, Anal. Chim. Acta 597, 163-170 (2007). 

56


PB 

Study Of In-Vitro Release Kinetics And Metabololites 

Of A Antidepressant Drug In Anionic Mceller Media- A 

New Approach For Nanotechnology In Medical Science 

R JAIN AND A PANDEY 

Department of Chemistry, Dr. Harisingh Gour University, Sagar, India 

Depression is estimated to affect nearly 340 million people worldwide and 18 million people in the 

US at any given time making it the third most costly and disabling illness in the US. Duloxetine 

hydrochloride is one of the main antidepressant used as a selective serotonin and norepinephrine 

reuptake inhibitor (SSNRI) for oral administration. As it works on central nervous system (CNS), 

drug should be more available to the cells. To increase the bioavailability and solubility drug can 

be formulated with SLS. In this work we propose and analyzed different approaches to study the 

in vitro kinetics dissolution behavior , major metabolites, and oxidation metabolic pathway in 

anionic miceller media with the help of Vanadium (V) as an oxidant. The drug obeyed the beers 

law over the range of 5-50µg/ml at λmax 230nm . 0.5% SLS in 6.8phasphate buffer is proposed 

good discriminative dissolution media for Duloxitine hydrochloride delayed release formulation. 

The quantization of drug release studied by high performance liquid chromatography.( HPLC). By 

fitting the dissolution data in various kinetic models find out delayed release formulation of 

Duloxitine hydrochloride obey the Higuchi model kinetics having linearity range 0.99 and release 

component ‘n’ obtain by the Korsmyer Pappas model indicate release mechanism followed by the 

anomalous diffusion process. As far as the oxidation of Duloxetine is concerns, no mechanistic 

studies have been published. In this study activation parameters for the oxidation of Duloxetine 

by V(V) in both aqueous and micellar medium have been proposed and Duloxetine epoxide is 

identified as a oxidation product of duloxetine. Its conformed by the LC/MS and 

Spectrophotometrically. 

Keywords: Delayed release, Duloxitine hydrochloride, Anionic Micelles, Vanadium, HPLC, 

LC/MS, Korsmyer Pappas model, UV Spectroscopy 

57


PB 

Pourous Nanoparticles formation using a Dendrimer 

Template 

L. BONACCORSI 1 , D. LOMBARDO 2 , A. LONGO 3 , E. PROVERBIO 1 

1. Dipartimento di Chimica Industriale e Ingegneria dei Materiali, 

Università Messina, Salita Sperone 31, S. Agata (Messina), I -98166, Italy 

2. CNR – IPCF, Istituto per i Processi Chimico Fisici - sez. Messina, C.da 

Papardo Salita Sperone, Messina, I-98158, Italy. 

3. CNR–ISMN, Istituto per lo studio dei Materiali Nanostrutturati - sez. 

Palermo, Via Ugo La Malfa 153, I-90146, Palermo, Italy 

Recently there has been increasing interest in the synthesis, characterization and processing of 

novel materials with controlled structural characteristics [1-5]. Particularly interesting in this 

respect is the construction of supra-molecular organic-inorganic nanostructured materials based 

on microporous and nanoporous materials [6]. We describe the self-assembly of a spherical 

complex driven by molecular recognition and the incorporation of alluminosilicate in a dendrimer 

charged surface. By using a carboxyl-terminated dendrimer species as a macromolecular template 

for zeolite formation, we detected the formation of porous, stable and nearly monodisperse 

spherical aggregates with an average radius of R=3500 Å. The condensed charge in the surface of 

the dendrimer, acting as the main driving force, influence the crystallites aggregation as well as the 

long-range assembly conditions for the zeolite growth. The main features of the self-assembly 

process has been characterised by means of Small Angle X-ray Scattering (SAXS), Scanning 

Electron Microscopy (SEM), X-ray Diffracton (XRD) and Energy Dispersive X-ray (EDX) 

microprobe analysis techniques. The main finding of our results suggest a possible mechanism for 

nano-aggregates formation based on a secondary aggregation process between (dendrimers-based) 

primary units. According to this view the growth of the zeolite on the dendrimer surface and the 

consequent screening of the surface charge promote the entanglement process between the 

primary units. This study put novel insight in the investigation of alternative protocols for the 

assembly mechanism of pourous materials [7, 8]. 

References 

[1] (a) C. J. Brinker, G. W. Scherrer, “Sol-Gel Science, The Physics and Chemistry of Sol-gel Processing”, Academic 

Press, San Diego (1990). 

[2].P. Judenstein and C. Sanchez, J. Mater. Chem., 6 (1996) 511-525. 

[3] Israelachvili, J. Intermolecular and Surface Forces. London: Academic; 1991. 

[4] D. Lombardo. Langmuir 25(5), 3271-3275, (2009). 

[5] (a) Lombardo, D.; Longo, A.; Darcy, R.; Mazzaglia, A. Langmuir 20, 1057, (2004). (b) D. Lombardo, N. Micali, V. 

Villari, M. Kiselev Phys. Rev. E, 70, 21402, (2004). (c) A. Mazzglia, N. Angelini, D. Lombardo, N. Micali, S. Patane, 

V. Villari, L. M. Scolaro J. Phys. Chem. B, 109 (15), 7258, (2005). 

[6] Velev, O. D.; Tessier, P. M.; Lenhoff, A. M.; Kaler, E. W. Nature, 401, 548, (1999) 

[7] Barrer R. M. Synthesis of zeolites. In: Zeolites. Drzaj B, Hocevar S, Pejovnik S (eds) Elsevier Science Publishers 

BV, Amsterdam (1985) 

[8] L. Bonaccorsi, D. Lombardo, A. Longo, E. Proverbio, A. Triolo. Macromolecules 42(2), 1239-1243 (2009). 

58


PB 

Surface enhanced raman spectroscopy for the direct 

detection of human thrombin interacting with the 

thrombin binding aptamer 

M. A. OCHSENKÜHN 1 AND C. J. CAMPBELL 1,2 

1. School of Chemistry, University of Edinburgh, Joseph Black Builidng, 

Kings Builidings, Edinburgh, EH9 3JJ,UK 

2. Division of Pathway Medicine, University of Edinburgh, 49 Little 

France Crescent, Edinburgh, EH16 4SB, UK 

Surface enhanced Raman spectroscopy (SERS) is developing into a powerful method for various 

sensor applications. The use of surface plasmon resonant nanoscale roughend nobel metal surfaces 

or particles allows Raman spectroscopy down to single molecules and provides signal strengths 

similar to fluorescence. Here we prove that it is possible to detect the interaction between human 

α thrombin and and the specific thrombin binding DNA aptamer (TBA). In our experiments we 

use AuroShells (Nanospectra Biosciences, Inc) nanoshells (NS) consisting of a dielectric silica 

core covered with a layer of gold which are designed for excitement with near infrared light 

(780 nm). The particles were self aggregated on aminopropyl triethoxy silaninzed glass slides for 

fixation. We functionalised the NS aggregates with the 5’-SH-TTTTTT modified 15 base TBA 

(GGTTGGTGTGGTTGG) hybridized to a hairpin loop structure[1]. As standard procedure 

preventing unspecific binding a SAM of mercaptohexanol was formed on the uncovered areas of 

the metal. SERS of the functionalized surface do not show strong signals, only weak and broad 

signals from the oligonucleotide. On incubation with thrombin strong signals appear at 822 cm -1, 

1130 cm -1 and 1540 cm -1 which can be assigned to different aromatic ring stretches and appear due 

to the G-quadruplex formation[2] of the aptamer. We suggest that the stacking of the guanine 

bases lead to a raised amount of order increasing the Raman cross-section and therefore amplifies 

certain stretching modes after the tertiary folding of the oligonucleotide. We also show that the 

used sample spot can be recovered with high signal reproducibility. Tests with extremely low 

target protein concentrations down to zeptomolar values, correlating to less then 10 molecules in 

the probing volume, also resulted in the same characteristic signals. In this case the interaction is 

only temporary and signal is lost after about 30 seconds but can be found at different timepoints of 

the experiment probably caused by diffusion of the target in equilibrium with the binding related 

g-quartet stabilisation. This simple approach for the detection of specific proteinbinding by SERS 

on NS can be used for highly sensitive sensing with the potential for intracellular live cell 

applications. 

References 

[1] Yao,W., et al., Biosens. Bioelectron. (2009), doi:10.1016/j.bios.2009.04.016. 

[2] Padmanabhan, K. and A. Tulinsky, Acta Crystallogr D Biol Crystallogr, 1996. 52(Pt 2): p. 272-82. 

59


PB 

Raman and SERS spectra of benzoic and related 

monocarboxylic aromatic acids 

M.R. LOPEZ-RAMIREZ, J.L. CASTRO, J.F. ARENAS AND J.C. OTERO 

Department of Physical Chemistry, Faculty of Science, 

University of Málaga, E-29071-Málaga, Spain. jc_otero@uma.es 

Benzoic acid is the simplest carboxylic acid derived from benzene and one of the most important 

from the chemical and biochemical points of view. Its many applications include synthesizing a 

large number of chemicals, food preservative and, together with salicylic acid, it is used in the 

treatment of fungal skin diseases. On the other hand, phenylacetic and 3-phenylpropionic acids 

have shown a broad range of biological activity. One of the aims of this work is to contribute to the 

research on the vibrational spectra of this series of molecules due to the lack of any complete 

vibrational study of these two related acids. Therefore the Raman spectra of benzoic, phenylacetic 

and 3-phenylpropionic acids have been assigned by applying the scaled quantum mechanical force 

field (SQMFF) methodology of Pulay et al. [1] to DFT force fields in order to fit the calculated 

frequencies to the experimental ones (Fig. 1). Given the agreement achieved between the computed 

and experimental frequencies, we think that the here obtained results provide a fairly good 

example of the usefulness of the SQMFF methodology to study the vibrational spectra of complex 

molecules. Furthermore, thanks to the huge potential of the experimental and theoretical 

approaches to modern surface science we can understand much better the properties of the ground 

electronic states of molecules adsorbed on solid surfaces. However, our knowledgment of the 

excited electronic states of adsorbed species, and the way they interact with the substrate is much 

lesser due to the relatively scarce number of techniques available to probe such states. Molecules 

adsorbed on some metal surfaces can exhibit enormously enhanced Raman scattering, and such 

technique has become known as Surface-Enhanced Raman Scattering (SERS) [2–4]. The SERS 

analysis methodology proposed by our group [5-6], is based on the use of ab initio calculations, and 

has shown its ability to explain in detail the observed experimental behavior and the differences 

between the SERS of these monocarboxylic aromatic acids, confirming so the relevance of the 

charge transfer mechanism in the enhancement of the Raman signal. 

References 

Fig. 1 – Transferring Scale factors between related acids (SQMFF). 

[1] W. L. Peticolas, D.P. Strommen, V.J. Lakshminarayanan, Chem. Phys. 73, 4185-4191 (1980). 

[2] M. Moskovits, Rev. Mod. Phys 57, 783-826 (1985). 

[3] B. Pettinger, “Adsorption of Molecules at Metal Electrodes” Edited by J. Lipkowski, P.N. Ross, VCH, New York, 285 

(1992). 

[4] A. Campion, P. Kambhampati, Chem. Soc. Rev. 27 241-250 (1998). 

[5] I. Lopez-Tocon, S. Centeno, J.L. Castro, M.R. Lopez-Ramirez, J.C. Otero, Chem. Phys. Lett. 377, 111-118 (2003). 

[6] J.F. Arenas, I.López-Tocón, J.L. Castro, S.P. Centeno, J.C. Otero, J. Raman Spectrosc. 36, 515-521 (2005). 

60


PB 

Talbot – Lau interferometry with bio-molecules and 

organic clusters 

M. SCLAFANI 1 , M. MARKSTEINER 1 , P. HASLINGER 1 , P. SCHMID 1 AND M. ARNDT 1 

1. Faculty of Physics, University Vienna, Quantum Optics, Quantum 

Nanophysics und Quantum Information Boltzmanngasse 5, 1090 Wien, 

Austria 

We discuss the prospects for Talbot - Lau quantum interferometry for testing the matter-wave 

duality of biological molecules and organo-metallic clusters. The quantum behaviour of large 

massive objects has already been successfully proved [1], but interferometry with organic 

molecules is still challenging, because of the required molecular beam and detection techniques for 

large neutral molecules. The present setup is designed for amino acids since they are often 

considered to be a model for larger bio-molecules such as peptides or proteins. Molecules are 

promoted into the gas phase by laser desorption into a supersonically expanding gas jet, and the 

individual particles are detected by VUV photo-ionization. We discuss the state of our experiment 

and we explore future applications of near-field matter wave interference [2] with regard to the 

determination of molecular polarizabilities, dipole moments or even conformations of small 

organic molecules. We further report on the production of large neutral tryptophan-metal 

complexes [3] as interesting objects for future matter wave experiments and on the first promising 

steps towards the detection of single neutral gas phase molecules using superconducting single 

photon detectors. 

References 

[1] M. Arndt, O. Nairz, J. Vos-Andreae, C. Keller, G. Van der Zouw and A. Zeilinger, Nature 401, (1999) 

[2] B. Brezger, M. Arndt and A. Zeilinger, J. Opt. B: Quantum Semiclass. Opt. 5, 82-89 (2003) 

[3] M. Marksteiner, P. Haslinger, H. Ulbricht, M. Sclafani, H. Oberhofer, C. Dellago and M. Arndt, J. Am. Mass 

Spectrom. 19-7, 1021-1026 (2008) 

61


PB 

Structure and stability of D-galactose/D-glucosebinding 

protein. The role of D-glucose binding and Ca 

ion depletion 

OLGA V. STEPANENKO 1 , O.I. POVAROVA 1 , OLESYA V. STEPANENKO 1 , A.V. FONIN 1 , I.M. 

KUZNETSOVA 1 , K.K. TUROVEROV 1 , M. STAIANO 2 , S.D’AURIA 2 

1 Institute of Cytology of the Russian Academy of Sciences, Tikhoretsky 

av. 4, Saint-Petersburg, 194064, Russia, 

2 Institute of Protein Biochemistry CNR, Via Pietro Castellino 111, 

Napoli, 80131, Italy 

Protein folding remains one of the most intriguing problems of molecular biology. When it seems 

that we were quite near the explanation of protein folding in compact globular structure it 

appeared that many proteins in principle cannot have such structure alone, without their partners 

(other proteins, DNA molecules, and inorganic ligand). In view of this new concept it is interesting 

to study the role of ligands in proteins structure and stability. The periplasmic binding proteins 

which can exist both in ligand-free and ligand-bound form seem to be an interesting candidate for 

such investigations. The aim of this work was the study of the structure, folding and stability of Dgalactose/D-glucose-binding 

protein (GGBP) from E. coli belonging to this protein family. GGBP 

molecule alongside with sugar has one more ligand – calcium ion localized in the loop of the Cterminal 

domain. Investigations were carried out by intrinsic and ANS fluorescence, far- and near- 

UV circular dichroism, scanning microcalorimetry. Parametric presentation of protein fluorescence 

intensity changes on the pathway of its unfolding were used to reveal the concealed intermediate 

states in the pathway of protein folding—unfolding if there is any. Kinetics of structural changes of 

GGBP and its complex with D-glucose (GGBP/Glc) induced by Ca depletion were recorded by the 

change of the intrinsic fluorescence intensity. Unfolding—refolding process of ligand-free and 

ligand-bound protein were induced by chemical agent guanidine hydrochloride (GdnHCl) and by 

heat, or both of them. It was shown that unfolding processes of both GGBP and its complex with 

D-glucose are one step reversible processes. Stability of GGBP and GGBP/Glc was evaluated in 

terms of the difference of free energy between native and unfolded state (ΔG). Calcium depletion 

results in 3D structure changes of GGBP. At the same time, the presence of bounded sugar makes 

GGBP more resistant to structure reorganization induced by calcium removal. Kinetic experiments 

have revealed that GGBP-Ca is less stable than GGBP while D-glucose binding increases GGBP- 

Ca/Glc resistance against denaturant. Stability of proteins is arranged as follows: GGBP-Ca < 

GGBP < GGBP-Ca/Glc < GGBP/Glc. The results of the work can have practical value as the 

differences in the structures of the ligand-bound and ligand-free proteins make GGBP good 

candidates for biological recognition element in the biosensor system for permanent noninvasive 

monitoring of glucose level in blood of diabetic patients. 

Acknowledgement: This work was supported by INTAS (YS 06-1000014-5586), NATO (CLG.983088), 

FASI (02.512.11.2277) and Program "Leading Scientific School of RF" (1961.2008.4). 

62


PB 

Using Nanodisks as a carrier for immobilization of 

membrane receptors in surface-enhanced infrared 

spectroscopy studies 

E. ZAITSEVA 1 , M.C. SAAVEDRA SAGREDO 1,2 , S. BANERJEE 3 , A. GRUNBECK 3 , T.P. SAKMAR 3 

AND R. VOGEL 3 

1. Institute of Molecular Medicine and Cell Research, University of 

Freiburg, Hermann-Herder Str. 9, Freiburg, 79104, Germany. 

2. Department of Physical Chemistry, Faculty of Chemical Sciences, 

University of Conception, Casilla 160-C, Concepcion, Chile. 

3. Laboratory of Molecular Biology and Biochemistry, 1230 York 

Avenue, New York, NY 10065, USA. 

G protein-coupled receptors (GPCRs) are involved in a broad range of physiological processes. 

Despite their crucial importance very little is known about the molecular mechanisms of signal 

transduction, bridging extracellular ligand binding with intracellular G protein activation. This is 

primarily due to the lack of techniques capable of following structural changes in GPCRs in the 

physiological lipid environment upon activation. Using surface-enhanced infrared spectroscopy 

(SEIRA), structure-function relationships of membrane receptors can be probed in a protein 

monolayer [1]. It is of importance to keep the receptors in a native-like environment to stabilize 

their functional structure. For SEIRA spectroscopy, this is a particular challenge as proximity of the 

receptor to the underlying gold surface has to be maintained in order to ensure sufficient infrared 

signal enhancement. On the other hand, destructive non-specific metal-protein interactions have to 

be avoided. An appealing possibility to achieve this goal is to reconstitute GPCRs into Nanodisks, 

consisting of a discoidal lipid bilayer encirculated by an apolipoprotein belt derived from 

lipoproteins [2]. Here we incorporate rhodopsin, a prototypical GPCR, into Nanodisks comprising 

zebrafisch apolipoprotein (zap-1) and palmitoyloleoyl phosphatidylcholine lipids (POPC). 

Nanodiscs with reconstituted rhodopsin were then tethered on a SEIRA-active gold surface while 

preserving function of the receptor. For SEIRA experiments nano-structured gold films deposited 

on the top of a trapezoid silicon prism were modified via thiol chemistry to produce a self 

assembled monolayer with nickel chelating nitrilo-triacetic acid (Ni-NTA) exposed to solution, and 

having a total thickness of about 3 nm. Loaded Nanodisks were then specifically bound to the 

surface using the his-tag of the zap-1. Formation of the monolayer of Nanodiscs on a SEIRA active 

surface was monitored using lipid and protein absorption bands. SEIRA difference spectroscopy 

was used to demonstrate that the immobilized receptor preserves its functional properties. In our 

experiments light-induced rhodopsin activation has resulted in reliably detectable SEIRA 

difference spectra, characteristic for the transition between the inactive and the signaling state 

Meta II. It can be concluded that Nanodisks is a suitable system to attach membrane proteins to a 

modified metal surface. They can be used to assess functional changes upon GPCR activation and 

ligand-protein interactions by means of vibrational spectroscopy. The results of this pilot study 

form a basis for investigations of other GPCRs activated by diffusible ligands. 

References 

[1] X. Jiang, E. Zaitseva, M. Schmidt, F. Siebert, M. Engelhard, R. Schlesinger, K. Ataka, R. Vogel, J. Heberle, Proc 

Natl Acad Sci USA. 105, 12113-7 (2008). 

[2] S. Banerjee, T. Huber, T.P. Sakmar, J.Mol.Biol. 377, 1067-1081 (2008). 

63

Protein dynamics 13 th ECSBM −−−− Book of Abstracts 

PB 

Theoretical study on correlation of geometrical 

structures and biological side effect in different drugs 

A. R. BEKHRADNIA AND M. A. EBRAHIMZADEH 

Dept. of Chemistry and Pharmaceutical Sciences Research Center 

Mazandaran, University of Medical Sciences, Sari, Iran 

Complete geometry optimizations were performed on diverse drugs with similar side effect. The 

considered drugs were selected apart from the more common biological effects. Frequency 

calculations and geometrical optimizations were carried out at the abinitio level of theory using the 

Gaussian software. The best conformers were found for these molecules and set up apparent 

pictures. These figures are along with thermodynamic data such as thermal energies (E), thermal 

enthalpies (H) and thermal Gibbs free energies (G) have also included in this study. All of the 

mentioned drugs were revealed that the structural requirements include an electron-rich 

functional group in the molecular plane of and separated from the center of an aromatic ring. In 

order to evaluate scope and investigation of these different drugs as their similar side effect (like 

hair-loss side effect), the geometrical center of the aromatic rings were served as the most easily 

defined reference point. The electron-rich functional group was considered in the plane of and 

separated from the aromatic ring center. For the best choice of electron-rich functional group and 

also a deeper investigation in considered drugs, it was used the calculated atomic charges. Then, 

the measurement of distance parameter between the related functional group and defined 

reference point was employed Gaussian program and its correlated softwares. These kinds of 

parameters are the most important factors in explanation of relationships in structure and 

reactivity and it is used to clarify some pharmacokinetic properties and biological effects. For 

drugs with hair-loss side effect, the calculated considered distances have had high consistency 

with 95% confidence limits (ca. 7.68 angstroms). Therefore, one can recognize that the related 

distance, is important and effective factor in structure of drugs with similar side effect. Therefore, 

one can recognize that the theoretical attempts are made to circumvent to biological side effect for 

the new synthesis in experimental study. 

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Thermal denaturation of myoglobin in water-sugar 

matrices and relationship with the glass transition of 

the system 

G. BELLAVIA 1 , G. COTTONE 1 , S. GIUFFRIDA 1 , L. CORDONE 1 AND A. CUPANE 1 



Glassy saccharide matrices protect proteins against denaturation under extreme environmental 

conditions as extreme drought, high or low temperature. The disaccharide trehalose is the best 

stabilizer largely used in nature [1]. The molecular mechanisms behind this effect are not yet fully 

understood and are, at present, matter of challenge. An hypothesis relates the stabilizing effects of 

disaccharides to the collective dynamic properties of the water/sugar matrix [2]. To further 

investigate this hypothesis we studied the relationship between the thermal denaturation (Tden) of 

a protein embedded in disaccharide/water and the glass transition temperature (Tg) of the system. 

To this end, we investigated by Differential Scanning Calorimetry the thermal denaturation of 

ferric myoglobin in water/sugar mixtures containing non–reducing (trehalose [3], sucrose) or 

reducing (maltose, lactose) disaccharides. All the samples studied are, at room temperature, liquid 

systems whose viscosity varies from very low to very large values depending on the water content. 

At high water/saccharide mole ratio, homogeneous glass formation does not occur; regions of 

glass form, whose Tg does not vary by varying the saccharide content, and the sugar barely affects 

the myoglobin denaturation temperature. At suitably low water/saccharide mole ratio, by 

lowering the temperature, the whole systems undergo transition to a glassy state whose Tg is 

determined by the water content; the Gordon Taylor relationship [4] between Tg and the 

water/disaccharide mole ratio is obeyed; Tden increases by decreasing the hydration regardless of 

the sugar, such effect being entropy driven. The presence of the protein found to lower the Tg 

respect to binary water-disaccharide systems [5]. Furthermore, for non–reducing sugars, plots of 

Tden vs. Tg give linear correlations, while for reducing sugars data exhibit an erratic behaviour 

below a critical water/sugar ratio, which depends on the particular disaccharide. We ascribe this 

behaviour to the likelihood that in the latter samples proteins have undergone Maillard reaction 

[6] before thermal denaturation. 

References 

[1] J.H. Crowe, Adv. in Experim. Med. and Biol., 594, 143, (2007). 

[2] J.L. Green, C.A. Angell, J. Phys. Chem., 93 (1989) 2880–82. 

[3] G. Bellavia, L. Cordone, A. Cupane, J. Therm. Anal. Calor., 95, (2009) 699–702. 

[4] M. Gordon, J.S. Taylor, J. Appl. Chem., 2 (1952) 493–500. 

[5] Y. Roos, J. Therm. Anal. Calor., 48 (1997) 535–544. 

[6] F. Ledl, E. Schleicher, Angewandte Chemie – Int. Ed., 29 (1990) 565–706. 

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TFE induced denaturation of YY1 protein 

P. BONAREK, A. GÓRECKI, E. KOWALCZYK, M. DZIEDZICKA-WASYLEWSKA 

Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian 

University, Gronostajowa 7 str., 30-387 Krakow, Poland 

2,2,2-Trifluoroethanol (TFE) stabilizes secondary structure formation in peptides and proteins 

therefore is widely used as a structure inducing cosolvent. Since we believe, that the human 

transcription factor belongs to family of intrinsically unstructured proteins, we investigated the 

TFE-induced denaturation of that protein. The circular dichroism and fluorescence spectroscopy 

studies indicate that the TFE-induced structural transition of YY1 proceeds via two distinct states, 

depending on TFE concetration. The experiments were performed using circular dichroism 

spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary 

structures, respectively. Size exclusion chromatography and dynamic light scattering were used to 

measure the hydrodynamic properties in different conformational states. 

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Dielectric properties of myoglobin at 10 GHz by 

microwave cavity perturbation measurements 

M. BONURA 1 , G. SCHIRÒ 1 AND A. CUPANE 1 



We report on the temperature dependence of the imaginary part of the dielectric constant (ε’’) in 

myoglobin (Mb) samples with different hydration (h= gr[H2O]/gr[Mb]). The measurements have 

been performed by the cavity perturbation technique, in the range of temperature 80÷345 K. The 

sample is located inside a NMR capillary along the axis of a cylindrical copper cavity, resonating in 

the TE011 mode at 9.6 GHz, where the electric field has a node. By measuring the variation of the 

quality factor of the resonant cavity, one can extract the imaginary part of the dielectric constant 

[1]. We have observed an evident increase of the dielectric energy losses on increasing the 

temperature from T~ 210 K; furthermore, the intensity of the variation is strongly dependent on 

the hydration. Since our working frequency is inside the region of dispersion of water molecules 

[2,3], our experimental technique allows us to investigate the role of hydration in protein 

dynamics. 

References 

Fig. 1 – Temperature dependence of the imaginary part of the 

dielectric constant in myoglobin-powder samples with different 

water content. 

[1] Z. Zai, C. Kusko, N. Hakim, S. Sridhar, A. Revcolevschi and A. Vietkine, Rev. Sci. Inst. 71(8), 3151-3160 (1991). 

[2] R. Buchner, J. Barthel, J. Stauber, Chem. Phys. Lett. 306, 57–63 (1999). 

[3] G. P. Singh, F. Parak, S. Hunklinger, K. Dransfeld, Phys. Rev. Lett. 47, 685-688 (1981). 

67


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Reaction mechanism of the GTPase Ran revealed by 

trFTIR studies utilizing isotopic labeling. 

S. BRUCKER, C. KÖTTING, K. GERWERT 

Department of Biophysics, Ruhr-University Bochum, Germany 

The GTPase Ran, member of the Ras super family, has a key role in eukaryotic cells during the 

nucleocytoplasmatic transport, regulates mitotic spindle and post mitotic nuclear assembly [1]. 

Ran is a molecular switch, characterized be the change between the GDP- and the GTP-bound 

form. Ran itself is up-regulated by guanine exchange factor (GEF) RCC1, down-regulated by 

GTPase-activating protein (GAP) Rna1p and is stabilized by Ran binding protein 1 (RanBP1). For 

the investigation of the reaction-mechanism by time resolved Fourier Transform Infrared 

Spectroscopy (trFTIR) isotopic labeled amino acids were incorporated into the protein [2]. The 

incorporation of the amino acids into the protein was done by optimized M9-media, the analysis of 

incorporation-rate and spreading of the label was done by mass spectrometric analysis. The 

bacteria cultures were optimized for the respective amino acid increasing incorporation and 

decreasing transformation of the label. Therefore also E. coli-strains auxotroph for the respective 

isotope-coded amino acid were used after optimization in consideration of growing and protein 

expression. By isotopic labeling accurate assignment of signals in trFTIR-spectroscopy by band 

shifts of the labeled groups contrary to the unlabeled ones is possible. Tyrosine 39 of switch I of 

Ran is located at the same position as the ‘argininefinger’ of Ras and coordinates glutamine 69 and 

the γ-phosphate [3]. During the interaction of Ran with its effectors in the GTP-bound form 

tyrosine 39 seals the binding pocket and excludes all waters other than the attacking water needed 

during hydrolysis, but at a misplaced position. It becomes positioned correctly when RanGAP 

binds and repositions glutamine 69. If tyrosine 39 is missing intrinsic hydrolysis gets faster, but for 

the GAP-catalyzed reaction tyrosine 39 has no influence. 

References 

Fig. 1 – 3D trFTIR spectrum of the GAP-catalyzed GTP hydrolysis of 

the Ran RanBP1 Rna1p complex (on the right). 

[1] M. Stewart (2007) Molecular mechanism of the nuclear protein import cycle, Nature Reviews Molecular Cell Biology 

8, 195-208. 

[2] B. Warscheid, S. Brucker, A. Kallenbach, H. E. Meyer, K. Gerwert, C. Kötting (2008) Systematic approach to 

group-specific isotopic labeling of proteins for vibrational spectroscopy, Vibrational Spectroscopy 48, 28-36. 

[3] M. J. Seewald, C. Körner, A. Wittinghofer, I. R. Vetter (2002) RanGAP mediates GTP hydrolysis without an arginine 

finger, Nature 415, 662-666. 

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Dynamics of interaction of hypericin with low-density 

lipoproteins 

D. BUZOVA1 , L. BURIANKOVA1 , D. BRAULT3 , D. CHORVAT JR. 2 , D. JANCURA1 , P. MISKOVSKY1, 2 

AND F. SUREAU3 1. Dept. of Biophysics, P.J. Safarik University, Kosice, Slovakia 

2. International Laser Center, Bratislava, Slovakia 

3. Univ. Paris 06, CNRS, Lab. Biophys. Mol. Cellulaire and Tissulaire, 

Evry, France 

Fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics 

of incorporation of hypericin (Hyp) into low-density lipoproteins (LDL). Hypericin, major 

component of the plants of the genus Hypericum, is a powerful naturally occurring photosensitizer 

with broad spectrum of light-induced biological activities [1, 2]. Upon administration into the 

blood stream Hyp associates with serum proteins, mainly with LDL [3], which are attractive 

vehicles for drug delivery and targeting [4]. Triphasic kinetics of Hyp association with LDL was 

observed when solutions of Hyp and LDL were mixed together. The most rapid phase of Hyp 

incorporation is completed within tens of msec, while the slowest one lasts 10-20 min. The most of 

Hyp molecules are incorporated into LDL in the slowest phase. The kinetics of the incorporation of 

Hyp into LDL particles pre-loaded with Hyp were also investigated. The observed decrease of the 

lifetime and total intensity of Hyp fluorescence with the increase of the incubation time of Hyp 

with Hyp/LDL complex is a sign of the formation of aggregates and the dynamic quenching of 

singlet excitation state of Hyp inside LDL [5]. To study the kinetics of a transfer of Hyp molecules 

between LDL particles, the time evolution of the stopped-flow and time-resolved fluorescence 

experiments were investigated after the mixing of the complex Hyp/LDL=200:1 with appropriate 

amounts of free LDL. For each final Hyp/LDL ratio the increase of the lifetime and total intensity 

of Hyp fluorescence was observed. The half-time of this process is similar to that one of the slowest 

phase of Hyp incorporation into free LDL. 

References 

[1] H. Falk, Angew. Chem. Int. Ed. Engl. 38, 3117-3136 (1999). 

[2] T. Kiesslich, B. Krammer, K. Plaetzer, Current Medical Chemistry 13, 2189-2204 (2006). 

[3] B. Chen, Y. Xu, T. Roskams, E. Delaey, P. Agostinis, J. R. Vandenheede, P. de Witte, Int. J. Cancer 93, 275-282 

(2001). 

[4] G. Jori, J. Photochem. Photobiol. B, 36, 87-93 (1996). 

[5] S. Kascakova, M. Refregiers, D. Jancura, F. Sureau, J.C. Maurizont, P. Miskovsky, Photochem. Photobiol. 81, 1395- 

1403 (2005). 

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CONFORMATIONAL ANALYSIS, FT-IR AND 

RAMAN SPECTROSCOPIC STUDY OF Cyclo(His-Phe) 

DIPEPTIDE 

SEFA CELIK 1 , AYSEN E. OZEL 2 , SEVIM AKYUZ 3 AND SERDA KECEL 2 

1. Istanbul University, Engineering Faculty, Electrical-Electronics 

Eng. Department, 34320-Avcilar, Istanbul, Turkey 

2. Istanbul University, Faculty of Science, Department of Physics, 

Vezneciler, 34134, Istanbul, Turkey 

3 Istanbul Kultur University, Faculty of Science and Letters, 

Department of Physics, Atakoy Campus, 34156 Istanbul, Turkey 

Theoretical conformational analysis method was applied to examine the three-dimensional 

structure and conformational properties of the cyclic dipeptides which have anticancer activity. 

Exploration of the conformational properties is necessary in order to understand the mechanism of 

the activity of a drug. In this study combined experimental study on molecular vibrations of 

cyclo(his-phe) has been investigated by molecular mechanics and ab-initio calculations. The 

calculations of cyclo(his-phe) dipeptide as a function of side chain torsion angles enable us to 

determine their energetically preferred conformations. The relative positions of the side chain 

residues of the stable conformations of dipeptide were obtained depending on the obtained 

conformational analysis results of using program proposed by Godjaev et al in FORTRAN[1]. The 

most stable conformation of the cyclo(his-phe) was found and the geometry were optimized using 

DFT method at B3LYP/6-31++G(d,p) level of theory. For computational studies Gaussian program 

were used.The vibrational normal modes and associated wavenumbers, IR intensities and Raman 

activities were calculated and the results were compared with these experimental data. The 

fundamental vibrational modes were assigned depending on their total energy distributions. 

References 

[1] N. M. Godjaev, I. S. Maksumov, I.J. L. I. Ismailova; J. Chem. Struc. (Russian) 24 147-152 (1983). 

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Structure and dynamics of Troponin I in thin filaments 

using SDSL-EPR 

J. COOKE 1 , J. CHAMOUN 2 , M. HOWELL 1 , P. FAJER 2 AND L. BROWN 1 

1. Dept. of Chemistry & Biomolecular Sciences, Macquarie 

University, Sydney, Australia 

2. Dept. of Molecular Biophysics, Florida State University, 

Tallahassee, FL, USA 

Troponin (Tn) is the molecular switch of striated muscle contraction. It is a heterotrimeric protein 

consisting of a calcium (Ca 2+) binding subunit (TnC), an inhibitory subunit (TnI), and a thinfilament 

anchoring subunit (TnT). To date, the proposed mechanism of the Ca 2+ dependent 

regulation of muscle by troponin is based mostly on data obtained in the absence of the thinfilament 

binding partners of Tn; tropomyosin and actin. Site Directed Spin Labeling Electron 

Paramagnetic Resonance (SDSL-EPR) has the advantage over other structural techniques as it 

enables the structural analysis of the Tn complex in the reconstituted thin filament. Several regions 

of the Tn complex are highly flexible causing difficulties with structural elucidation using 

traditional high-resolution techniques. One such important region is the C-terminal region of the 

Tn inhibitory subunit (C-TnI), a proposed secondary actin-binding domain. Several contradicting 

structural models of C-TnI have been suggested, all in the absence of actin, with most describing a 

highly dynamic domain with some secondary structural content [1-2]. Methane thiosulfonate spin 

labels (MTSSL) were introduced at cysteine residues in the cardiac isoform of TnI (residues 

Cys175-206). The mobility trends across the C-terminus displayed a weak helical interaction with 

actin in the reconstituted thin filament. Interspin distances for three double cysteine mutant pairs 

(Cys176/178, Cys176/179 & Cys176/180), as measured by CW-EPR, correlated with this helical 

structure. An additional double cysteine mutant pair (Cys178/206) further confirmed an extended 

structure with an interspin distance of 34 Å on interaction with the thin filament. Key actinbinding 

residues, as identified by the EPR probe-mobility measurements, correlated with a 

number of C-TnI genetic mutations implicated in cardiomyopathy. A second region of interest is 

the ‘switch peptide’ of TnI which, upon Ca 2+ binding to TnC, is proposed to undergo a large-scale 

movement, allowing for muscle contraction (‘ON’ state) to occur. In this ‘ON’ state, the switch 

peptide (residues 150-159) is believed to be in close proximity to the N-lobe of TnC. In the absence 

of Ca 2+ (‘OFF’ state), the switch peptide is then released from TnC to inhibit the interaction of 

myosin with the actin thin filament. The proposed mechanism of this Ca 2+ dependent regulation is 

based on static x-ray structures of the Tn core regions of the skeletal isoform, also obtained in the 

absence of the thin filament [2]. Two spin-labeled cys mutant pairs were designed to monitor the 

proposed movement of the switch peptide in the reconstituted thin filament 

(TnI Cys151/TnC Cys35 & TnI Cys151/TnC Cys84). Distances were measured using both CW and 

pulsed EPR methods (DEER). The switch peptide was indeed tightly bound to the N-domain of 

TnC in the ‘ON’ state with short distances with narrow distributions, while unbound in the ‘OFF’ 

state with long distances and broad distributions. Further, the interaction of Tn with the actin thin 

filament appears necessary for the complete release of the TnI switch region from TnC. 

References 

[1] K. Murakami, et al, J Mol Biol 352, 178 (2005) 

[2] T. M. Blumenschein, et al, Biophys J. 90, 2436 (2006) 

[3] M. V. Vinogradova, et al , PNAS 102. 5038 (2005) 

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INFLUENCE OF ACETYL-D-GLUCOSAMMINE 

(NAG) INHIBITOR ON LOW FREQUENCY 

DYNAMICS OF LYSOZYME 

C. CRUPI 1 , G. D’ANGELO 1 , U. WANDERLINGH 1 , V. CONTI NIBALI 1 AND C. VASI 2 

1. Dept. of Physics, University of Messina, C.da Papardo - Salita Sperone 

31, 98166 Messina, Italy 

2. IPCF-CNR, Messina, C.da Papardo - Salita Sperone - 98158 Faro 

Superiore Messina, Italy 

The verified connection between biological activity of proteins and their conformational flexibility 

has fomented a growing interest just in low-frequency vibrations, in the THz range. It is believed 

that these modes are particularly sensitive to the specific conformation of the protein and that they 

drive the pathways to functionally important conformational transitions in proteins [1]. The 

occurrence of these low frequency motions in proteins is clearly observed in Raman spectra, below 

300 cm -1, where the presence of an asymmetric peak, commonly named Boson Peak, discloses the 

existence of an excess of vibrational density of states with respect to the Debye regime. Quite 

interestingly, a similar excess of low frequency vibrations over the Debye model prediction is also 

revealed in the specific heat, Cp, below 30 K, where, once more, it appears as a peak in the reduced 

Cp(T)/T 3 plot [2]. At present, even though there is a general consensus that these motions have to 

involve either all or a very large portion of the whole protein molecule [3,4,5] their origin and their 

precise biological role is still missing. In order to gain insights into the mysterious mechanisms 

which give rise to this excess of modes, the study of changes in low frequency vibrational 

dynamics of a protein following its interaction with a ligand and with and without the presence of 

water, would be really enlightening. With this aim, Raman scattering (at room temperature) and 

low temperature specific heat measurements have been performed on both native hen egg white 

lysozyme (HEL) and lysozyme including the acetyl-D-glucosammine (HEL/NAG) substrate at two 

different hydration degrees (without water and hydrated at h=0.35g water/g HEL or HEL/NAG). 

Light scattering results in agreement with calorimetric findings have shown that, the presence of 

the inhibitor strongly reduces the intensity of the Boson Peak while, upon hydration, in addition to 

this decreasing number of modes, the bump position shifts toward a higher frequency (or 

temperature). 

References 

[ 1 ] Berendsen, H. J. C., S. Hayward, Curr. Opin. Struct. Biol. 10, 165–169 (2000); 

[ 2 ] D'Angelo G. et al. unpublished data; 

[ 3 ] Joti Y., Nakagawa H., Kataoka M., Kitao A., Biophys. J. 94, 4435-4443 (2008); 

[ 4 ] Tarek M., Tobias D.J., J. Chem. Phys. 115, 1607-1612 (2001); 

[ 5 ] Chou K.C., Biophys. Chem. 25, 105 (1986). 

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Molecular modeling of the S-S bridges reduction in 

proteins 

C. DAVID 1 AND M. ENESCU 1 

1. Laboratoire de Chimie Physique et Rayonnement -Alain Chambaudet, 

Université de Franche Comté UMR CEA E4, 16, route de Gray, 25030 

Besançon CEDEX, France 

The native disulfide bridges (S–S) are considered to be a very important key in the folding and 

structure stabilisation of many proteins. The importance of the study of the protein disulfide 

bridges reduction comes to light in protein unfolding/refolding experiments. [1] The protein 

structure plays a significant role in the reduction mechanism.. It affects the reactivity of the 

bridges by three factors : the electrostatic interactions between reagents and the charged 

environment, the accessibility of the reaction site and the stabilisation of the reaction products by 

subsequent protein unfolding. The molecular modeling methods provide important information 

about this mechanism. We report here a systematic theoretical study on the reactivity of the four 

lysozyme S–S bridges. The bridges accessibility with respect to the reducing reagent TCEP is 

characterized by calculating QM/MM (PM3/Amber) potentials of mean force (PMF) curves. [2] 

The quantum mechanics-free energy perturbation (QM-FEP) method [3] was then employed for 

obtaining reaction free energy profiles. In this approach the chemical system configurations and 

point charges on the reaction trajectory were derived from QM (DFT) calculations on model 

systems. They were then used in classical dynamic simulations where only the changes in the 

reaction environment were sampled. Significant differences in the lysozyme S–S bridges’ reactivity 

were found (with a maximum for the cys6-cys127 bridge) thus proving the role of the protein 

structure in this reaction. 

References 

Fig. 1 –Free energy profiles for the reduction of a S-S bridge 

model. 

[1] W. J. Wedemeyer, E. Welker, M. Narayan, H. A. Scheraga, Biochemistry, 39, 4207-4216 (2000). 

[2] B. Roux, Comput. Phys. Commun., 91, 275-282 (1995). 

[3] J. J. Ruiz-Perna, E. Silla, I. Tun, S. Mart, V. Moliner, J. Phys. Chem. B, 108, 8427-8433 (2004). 

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Microstructure at the water lipid protein interface 

controls conformational switching mechanisms in the 

conserved D(E)RY motif of G-protein coupled receptors 

S. EICHLER , S. MADATHIL AND K.FAHMY 

Dept. of Biophysics, Institute of Radiochemistry, Forschungszentrum 

Dresden-Rossendorf, PF 510119, Dresden, D-01314, Germany 

G-Protein coupled receptors (GPCRs) play a fundamental role in many physiological processes 

due to their ability to switch between different structures upon activation. The prototypical GPCR 

rhodopsin serves as a model to study molecular switching mechanisms. Upon photoisomerization 

of the chromophore retinal, protonation of a glutamic acid (Glu 134) in the highly conserved 

D(E)RY motif at the cytosolic end of transmembrane helix 3 (TM3) leads to breakage of an ionic 

lock which stabilizes the inactive state [1]. Due to the low dielectricity of the lipidic environment, 

side chain charges and their neutralization contribute to the energetics of conformational 

transitions much more than in a purely aqueous environment. Our aim is to elucidate the 

functional implication of lipid protein interactions and microstructure at the water lipid protein 

interface in controlling protein conformation. We have studied synthetic peptides derived from 

rhodopsin TM3 by fluorescence spectroscopy at different pH in a hydrophobic environment. In [2] 

pH dependency of FRET between Trp at the cytosolic side of a TM3-derived peptide and 

DANSYL-PE was used as a monitor for helix motion. The observed pH dependency argues for 

stabilization of the protonated state by lipid protein interactions. In addition, we studied a TM3derived 

peptide with a Trp probe shifted into the hydrophobic region. This peptide showed a redshifted 

emission maximum of Trp, indicative of water accessibility. Moreover, at low pH the redshift 

was less pronounced supporting the hypothesis that the neutralized Glu134 repels water and 

in general provides a pH-regulated hydration site. We conclude that microstructure at the water 

lipid protein interface and lipid protein interaction play a key role in the switching mechanism of 

GPCRs. The predominance of these local interactions which are not strictly dependent on 

intramolecular contacts to specific amino acids reconciles the highly conserved proton uptake at 

the D(E)RY motif in GPCR activation on the one hand and the diverse ligand specificity of class-A 

GPCRs on the other hand. 

References 

[1] J. A. Ballesteros, A. D. Jensen, G. Liapakis, S. G.F. Rasmussen, L. Shi, U. Gether, J. A. Javitch, J. Biol. Chem. 276, 

29171-29177 (2001). 

[2] S. Madathil, G. Furlinski, K.Fahmy, Biopolymers 82, 329-333 (2006). 

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Time-Resolved Surface Enhanced Resonance Raman 

Spectro-Electrochemistry of Heme Proteins 

M. GROßERÜSCHKAMP 1, 2 , C. NOWAK 1, 2 , W. KNOLL 2 AND R.L.C. NAUMANN 1 

1. Max Planck Institute for Polymer Research, Ackermannweg 10, 

55128 Mainz, Germany 

2. Austrian Research Centers GmbH - ARC, Donau-City-Straße 1, 

1220 Wien, Austria 

The major part of the energy requirement of the human body is provided by the respiratory chain 

in form of adenosine triphosphate (ATP) by the respiratory chain. The multi redox center protein 

Cytochrome C Oxidase (CcO) plays a key role in this process [1]. Four coupled redox centers, CuA, 

heme a, heme a3 and CuB, are involved in the catalytic turnover of the protein. Surface enhanced 

resonance Raman spectroscopy (SERRS) combined with electrochemical methods is an adequate 

tool to study the redox processes of heme proteins such as CcO [2]. In contrast to CcO the heme 

protein cytochrome c exhibits only one heme structure. It was used as a benchmark system to 

develop a novel SERRS active substrate based on Ag nanoparticles as well as a measuring cell 

design allowing for high sensitive time-resolved SERRS measurements [3]. 

References 

Fig. 1 – Cytochrome c adsorbed on a self assembled monolayer of 

mercaptoethanol on a SER(R)S-active silver substrate (left) and the 

SERR spectra resulting from a potentiostatic titration of the protein 

(right). 

[1] M.K.F. Wikström, Nature 266, 271-273 (1977). 

[2] S. Lecomte, H. Wackerbarth, T. Soulimane, G. Buse, P. Hildebrandt, J. Am. Chem. Soc., 120, 7381-7382 (1998). 

[3] M.Grosserueschkamp, M. Friedrich, M. Plum, W.Knoll, R.L.C. Naumann, J. Phys. Chem B, 113, 2492-2497 (2009). 

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Protein diffusion in crowded electrolyte solutions 

M. HENNIG 1,2 , F. ROOSEN-RUNGE 1,2 , T. SEYDEL 1 , F. ZHANG 2 , M. W. A. SKODA 3 , R. M. J. JACOBS 4 , 

S. ZORN 1 , M. MACCARINI 1 , P. FOUQUET 1 AND F. SCHREIBER 2 

1. Institut Laue-Langevin, 6 Rue Jules Horowitz, Grenoble, F-38000, 

France 

2. Dept. of Applied Physics, University of Tuebingen, Auf der 

Morgenstelle 10, Tuebingen, D-72076, Germany 

3. ISIS, Rutherford Appleton Laboratory, Chilton, Didcot, UK 

4. Chemistry Research Laboratory, University of Oxford, Oxford, UK 

In a simplified picture, living cells operate through the motion of proteins embedded in a 

"crowded" aqueous solution of various macromolecules and salts [1]. Considerable debate 

therefore addresses the connection of protein function and protein motion in an aqueous 

environment as a function of environmental parameters such as charges and temperature. It can be 

assumed that protein function cannot be understood without taking into account protein motion in 

an aqueous environment and the interaction of proteins, ions, and water [2]. Neutron spectroscopy 

has since long been proven to be a unique tool to investigate protein dynamics [3]. Neutrons probe 

motion as a function of length scale from interatomic to mesoscopic distances in the sample, on 

time scales from sub-picoseconds to approximately 200 nanoseconds. Most of the early neutron 

spectroscopy work concerning protein dynamics has been performed on powders or hydrated 

powder samples [4]. Interestingly, the biologically highly relevant case of protein solutions has 

rarely been studied, inter alia due to the difficulty of discriminating the contribution from the 

centre-of-mass diffusion of both protein and solvent molecules and also the problem of obtaining 

the required protein scattering volume whilst keeping the solvent scattering contribution low. We 

present a combined cold neutron backscattering and spin-echo study of the self-diffusive and 

collective center of mass diffusion of the model globular protein Bovine Serum Albumin (BSA) 

under the condition of ”protein crowding” in aqueous solution. We investigate the protein 

diffusion on the nanosecond timescale as a function of protein volume fraction, ionic strength and 

temperature. Complementary, small-angle X-ray scattering data are used to obtain information on 

the correlations of BSA molecules in solution. Particular emphasise is put on the effect of 

crowding, i.e. conditions under which the proteins cannot be considered as objects independent of 

each other. We thus address the question at which concentration this crowding starts to influence 

the static and especially the dynamical behavior. The interpretation of the data is put in the context 

of existing studies on related systems and of existing theoretical models. 

References 

[1] R. Ellis, Macromolecular crowding: an important but neglected aspect of the intracellular environment, Current 

Opinion in Structural Biology 11 (1) (2001) 114-119. 

[2] P. Ball, Water as an active constituent in cell biology, Chem. Rev 108 (1) (2008) 74-108. 

[3] M. Bee, Localized and long-range diffusion in condensed matter: state of the art of QENS studies and future 

prospects, Chemical Physics 292 (2-3) (2003) 121-141. 

[4] F. Gabel, D. Bicout, U. Lehnert, M. Tehei, M. Weik, G. Zaccai, Protein dynamics studied by neutron scattering, 

Quarterly Reviews of Biophysics 35 (04) (2003) 327-367. 

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Structure differences between protein crystals and 

solution structures monitored by Raman spectroscopy 

V. KOPECKÝ JR. 1 , K. HOFBAUEROVÁ 1,2 , J. KOHOUTOVÁ 3 , J. ŠTĚPÁNEK 1 AND R. ETTRICH 3 


University in Prague, Ke Karlovu 5, Prague 2, CZ-121 16, Czech Republic 

2. Institute of Microbiology, Academy of Sciences of the Czech Republic, 

Vídeňská 1083, Prague 4, CZ-142 20, Czech Republic 

3. Institute of Systems Biology and Ecology, Academy of Sciences of the 

Czech Republic, Zámek 136, Nové Hrady, CZ-373 33 Czech Republic 

Raman spectroscopy gives a unique opportunity to study protein samples in different phases. It is 

possible to measure intact protein crystals directly in hanging drops in crystallization boxes were 

they growth and to study the protein structure in crystals as well as chemical reactions in single 

crystals [1]. Moreover, we apply a new technique of Raman spectroscopy – a drop coating 

deposition Raman (DCDR) method [2], based on a coffee ring effect, that enables measurements of 

solutions down to 1 µM concentrations. However, our recent work adverted to subtle differences 

which correspond to the glass-like phase of the deposited samples [3]. Thus, DCDR protein 

samples represent a "phase transition" between oversaturated protein solutions and crystals. This 

enables to distinguish spectral differences given by the density of molecules in crystals from those 

caused by protein crystal artifacts. Here we illustrate, on several examples, applicability of the 

method for distinguishing these differences and improving X-ray protein structures by molecular 

modeling based on information from Raman spectroscopy. 

Fig. 1 – I) Crystals of PsbP protein; II) DCDR ring of PsbP protein; III) Raman spectra of 

PsbP protein measured in (A) solvent, as (B) DCDR deposit and in (C) the crystal form. 

Appropriate spectral differences are depicted down the figure. 

References 

[1] P. R. Carey, J. Dong, Biochemistry 43, 8885–8893 (2004). 

[2] D. Zhang, Y. Xie, M. F. Mrozek, C. Ortiz, V. J. Davisson, D. Ben-Amotz, Anal. Chem. 75, 5703–5709 (2003). 

[3] J. Kapitán J., V. Baumruk, V. Kopecký Jr., R. Pohl, P. Bouř, J. Am. Chem. Soc. 128, 13451–13462 (2006). 

77


PB 

Volume fluctuations in solvated protein by ultrasonic 

spectroscopy and molecular dynamics simulation 

T. HUSHCHA 1 AND S. YESYLEVSKYY 2 

1. Institute of Bioorganic Chemistry and Petrochemistry, National 

Academy of Sciences of Ukraine, Murmanska str.1, Kyiv – 94, 02660, 

Ukraine 

2. Institute of Physics, National Academy of Sciences of Ukraine, 

Prospect Nauki 46, Kyiv, 03039, Ukraine 

Constant pressure molecular dynamics techniques have been employed to investigate the changes 

in structure and volume of globular protein, human serum albumin, on the time scale of 100 ns. 

Computed apparent volume and compressibility of protein are close to experimental values that 

have been also estimated in this paper by density and sound velocity measurements. Relaxation of 

apparent protein volume after external pressure change has been investigated by molecular 

dynamics simulation that mimics the broadband ultrasonic spectroscopy measurements 

performed in our previous paper [1]. The structural effects observed in the simulations can well 

explain protein compressibility measurements carried out by ultrasonic technique. We observe 

that the protein apparent volume relaxes involving two time scales: a slower one with the 

characteristic time close to 100 ns due probably to conformational changes in protein chain and a 

faster one exhibiting distribution of relaxation times around 0.1 ns. The latter can be attributed to 

protein-solvent interactions. We find that two phenomena give unequivalent contributions to 

fluctuations of the protein apparent volume. The total fluctuations resulting from hydration 

changes significantly exceed those resulting from the protein intrinsic motions. It is interesting that 

the principal component analysis of molecular dynamics trajectories shows that the main 

contribution to protein intrinsic volume fluctuations comes from low frequency modes related to 

large and slow motions of proteins [2]. We conclude that even within the 100 ns time scale intrinsic 

volume fluctuations of protein do not exceed its partial volume changes resulting from hydration 

rearrangements. 

References 

[1] T. Hushcha, U. Kaatze, A. Peytcheva, Biopolymers 74, 32-36 (2004). 

[2] F. Tama, O. Miyashita, A. Kitao, N. Go, Eur. Biophys. J. 29, 472-480 (2000). 

78


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CONFORMATIONAL ANALYSIS AND VIBRATIONAL 

SPECTROSCOPIC INVESTIGATION OF L-ALANYL-L- 

GLUTAMINE DIPEPTIDE 

SERDA KECEL 1 , AYSEN E. OZEL 1 , SEVIM AKYUZ 2 AND SEFA CELIK 3 

1. Istanbul University, Faculty of Science, Department of Physics, 

Vezneciler, 34134, Istanbul, Turkey 

2 Istanbul Kultur University, Faculty of Science and Letters, 

Department of Physics, Atakoy Campus, 34156 Istanbul, Turkey 

1. Istanbul University, Engineering Faculty, Electrical-Electronics Eng. 

Department, 34320-Avcilar, Istanbul, Turkey 

Glutamine is the most abundant amino acid in the human body, and is the important intermediate 

of nitrogen metabolism. It was shown that surgical patients greatly benefit from supplementary 

glutamine dipeptides and glutamine dipeptides help to reduce the infectious complications [1]. 

Glutamine is also found to prevent chemotherapy and radiation-induced toxicity [2]. L-alanyl-Lglutamine 

(Ala-Gln) dipeptide helps to diminish tumor growth by restoring the function of natural 

killer cells increasing selectivity of antitumor drugs [3]. In spite of its great biological importance 

there is no study on experimental and theoretical vibrational spectra of Ala-Gln dipeptide was 

reported. In the first part of this study the conformational behavior of Ala-Gln dipeptide has been 

investigated by molecular mechanic and ab-initio calculations. The energy calculations of Ala-Gln 

dipeptide as a function of side chain torsion angles enable us to determine their energetically 

preferred conformations. The relative positions of the side chain residues of the stable 

conformations of dipeptide were obtained depending on the obtained conformational analysis 

results of using program proposed by Godjaev et al in FORTRAN. The lowest energy conformer 

has been determinate by using the Ramachandran maps and compared with the quantum 

chemical ab-initio results. The geometry optimization, vibrational wavenumber and intensity 

calculations of Ala-Gln dipeptide were carried out with the Gaussian03 program by using DFT 

with B3LYP functional and 6-31++G(d,p) basis set. The IR spectra (4000-400cm-1) and micro 

Raman spectra of the la-Gln dipeptide, in solid phase, have been reported and compared with the 

theoretical vibrational data. 

References 

[1] N. M. Godjaev, I. S. Maksumov, I.J. L. I. Ismailova; J. Chem. Struc. (Russian) 24 147-152 (1983). 

79


PB 

Non-trivial quantum effects in radical-ion-pair 

reactions 

I. KOMINIS 

Dept. of Physics, University of Crete, Heraklion 71103, Greece and 

Institute of Electronic Structure and Laser, Foundation for Research and 

Technology, Heraklion 71110, Greece 

Non-trivial quantum effects in biology have been sought after for a long time. It was recently 

discovered that a familiar biological system, namely radical-ion pairs and their reactions, exhibits a 

number of quantum effects until now manifested only in carefully prepared quantum physics 

experiments with well-isolated atomic systems. Radical-ion-pairs are bio-molecules fundamental 

in the photosynthetic reaction dynamics, are understood to underlie the chemical compass used by 

avian species for navigation and are found in several other biochemical contexts. It has been 

shown that magnetic-sensitive radical-ion-pair reactions sustain quantum coherence, exhibit 

quantum jumps and the quantum Zeno effect and are the first biological system where the full 

machinery of quantum measurement theory can be fruitfully applied. This opens the way to 

several vistas in theoretical and experimental quantum biology, from the understanding of the 

fundamental connections between quantum-dynamic effects and biology, to the design and 

experimental demonstration of novel quantum-limited biochemical-reaction magnetometers and 

last but not least to the exploration of quantum information processing at the biological level. 

References 

[1] I. K. Kominis, http://aps.arxiv.org/abs/0806.0739 

80


PB 

Ultrafast spectroscopy of photoactive flavoproteins 

A. LUKACS 1,2 , M. H. VOS 2 AND S. R. MEECH 1 

1. Faculty of Chemical Sciences and Pharmacy, University of East 

Anglia, United Kingdom 

2. Laboratoire d’Optique et Biosciences, Ecole Polytechnique, France 

More than a hundred enzymes exist which use the flavin chromophore as a cofactor. Flavoproteins 

exhibit a rich redox chemistry as the flavin chromophore can adopt three redox states, oxidized, 

one electron reduced (semi-reduced), two electron reduced (fully reduced). The different forms of 

the flavin chromophore have characteristic absorption spectra in the visible and near UV, but the 

physiological functions of most flavoproteins are light independent. A special class of 

flavoproteins comprises those proteins where the redox process is initiated by light. This specific 

property makes photoactive flavoproteins, phothotropins, photolyases, cryptochromes, and BLUF 

(blue light sensing using FAD) proteins suitable for investigation with laser spectroscopy, where 

the laser flash triggers the photochemical process. In this study we present experiments made on 

photolyase and AppA. Photolyase is a photoactive flavoprotein where the absorption of blue light 

results in repair of UV induced DNA lesions, via an electron transfer process from the fully 

reduced flavin cofactor (FADH–) to the substrate [1]. Ultrafast transient absorption measurements 

were performed on different mutant photolyases in order to describe the photoreduction [2] 

process in photolyase which converts the semi-reduced flavin chromophore in its catalytically 

active (fully reduced) form. We also present ultrafast time-resolved infrared (TRIR) studies of a 

BLUF containing flavoprotein, the transcriptional antirepressor AppA in which the formation of 

the light-induced signaling state is thought to be a result of multi-step electron and proton transfer 

processes [3,4]. Light and dark adapted forms of the protein are investigated by ultrafast TRIR and 

isotopic substitution.[5] 

Fig. 1 – A) p53 labeled with 4-aminothiophenol (4-ATP)-Np SERS marker; B) p53-Az biorecognition 

event. 

References 

[1] N. L. Rosi and C. A. Mirkin. Chemical Reviews 105 (2005) 1547. 

[2] A. Otto, I. Mrozek, H. Grabhorn, and Y. Pommier, J. Phys.: Condens. Matter 4 (1992) 1143. 

[3] A. R. Bizzarri, S. Cannistraro, Nanomedicine 3 (2007) 306. 

[4] M. Taranta, A. R. Bizzarri and S. Cannistraro, J. Mol. Recognit. 21 (2008) 63. 

81


PB 

Spectroelectrochemical study of [NiFe] hydrogenase of 

Desulfovibrio vulgaris Miyazaki F in solution and 

immobilized on biocompatible gold surfaces 

D. MILLO 1 , M. E. PANDELIA 2 , T. UTESCH 1 , N. WISITRUANGSAKUL 1 , M. A. MROGINSKI 1 , W. 

LUBITZ 2 , F. LENDZIAN 1 , P. HILDEBRANDT 1 AND I. ZEBGER 1 

1. Institut für Chemie, Technische Universität Berlin, Str. des 17. Juni 

135, Sekr. PC14, D10623-Berlin, Germany 

2. Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, D- 

45470 Mülheim/Ruhr, Germany 

The [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F was immobilized at biocompatible 

Au electrodes and studied by surface-enhanced infrared absorption (SEIRA) spectroscopy in 

combination with direct electrochemistry. The advantage of this approach is that electrochemical 

investigations, such as cyclic voltammetry (CV), can benefit from the unique structural insight 

given by SEIRA. In fact, in the case of hydrogenase, SEIRA spectroscopy provides information 

about the secondary structure of the protein backbone (in the spectral region between 1500 and 

1700 cm -1) and about the redox state of the catalytic active site as reflected by the stretching modes 

of the CO and CN - ligands (in the spectral region between 1900 and 2100 cm -1). [1] Comparison 

between IR spectra of the enzyme obtained in solution [2] with those obtained on the surface 

(SEIRA) reveals that protein immobilization does not alter the active site structure. Moreover, after 

immobilization, the adsorbed enzyme undergoes a slow reductive activation under H2, followed 

by a fast inactivation under O2 gas exchange, as observed in solution. Protein film voltammetry 

(PFV) proved that the adsorbed enzyme is catalytically active and undergoes reversible anaerobic 

inactivation upon increasing the applied potential. The reductive activation occurs at a potential 

(Eswitch) of -33 mV (vs NHE). SEIRA measurements in combination with PFV revealed that 

applying a potential to the electrode leads to a slow but steady irreversible decrease of the catalytic 

activity of the immobilized enzyme. The studies aim to gain deeper insight into the mechanism of 

the catalytic cycle of the [NiFe] hydrogenase as well as into protein-surface interactions, and may 

contribute to establish a novel methodology for studying interfacial biocatalytic processes. 

References 

[1] N. Wisitruangsakul, O. Lenz, M. Ludwig, B. Friedrich, F. Lendzian, P. Hildebrandt, I. Zebger, Angew. Chem 48, 

611-613 (2009). 

[2] C. Fichtner, C. Laurich, E. Bothe, W. Lubitz, Biochemistry 45, 9706-9716 (2006). 

82


PB 

Two Conformations of the Cytochrome c Oxidase 

Discriminated by Spectro-Electrochemistry Using 

SEIRAS 

CHRISTOPH NOWAK 1, 3 , JIAPENG ZHU 2 , ROBERT B. GENNIS 2 , WOLFGANG KNOLL 3 , RENATE L. C. 

NAUMANN 1 

1 Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 

Mainz, Germany 

2 University of Illinois, Department of Biochemistry, 600 South Mathews 

Street, Urbana, IL 61801, USA 

3 Austrian Research Centers GmbH, ARC, Donau-City Str. 1, 1220 

Vienna, Austria 

Electronic wiring of cytochrome c oxidase (CcO) from R. sphaeroides to gold surfaces was employed 

to monitor redox changes through redox centers, CuA, heme a, heme a3 and CuB. Electrochemical 

investigations revealed that under aerobic and reducing conditions the enzyme undergoes a 

gradual transition into an activated state. It is only in this state that proton pumping and catalytic 

currents can be observed. The potential of the catalytic current, however, is shifted by 450 mV 

negative from the standard redox potential of CuA. In contrast, “correct” standard redox potentials 

of all the centers in the positive potential range can be observed if the enzyme kept under 

anaerobic and oxidizing conditions. Then no proton pumping does take place. This state is 

therefore considered as a non-activated state. The transition between the two states is fully 

reversible. This was also verified by electrochemically-controlled surface-enhanced infrared 

absorption spectroscopy (SEIRAS). 

absorbance / a.u. 

9.0x10 -3 

8.0x10 -3 

7.0x10 -3 

6.0x10 -3 

5.0x10 -3 

4.0x10 -3 

3.0x10 -3 

2.0x10 -3 

1.0x10 -3 

0.0 

References 

ACTIVATED NON-ACTIVTED 

1700 

1654 

1682 1638 

1623 

2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 

wavenumber / cm -1 

1600 

1435 

A 

[1] Ch. Nowak, Ch. Luening, W. Knoll, R. L. C. Naumann, A two-layer gold surface with improved surfaceenhancement 

for spectro-electrochemistry using SEIRAS, Appl. Spec. under review 

83 

absorbance / a.u. 

9.0x10 -3 

8.0x10 -3 

7.0x10 -3 

6.0x10 -3 

5.0x10 -3 

4.0x10 -3 

3.0x10 -3 

2.0x10 -3 

1.0x10 -3 

0.0 

1656 

1639 

1681 1620 

1607 

1435 

B 

2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 

wavenumbe in cm -1


PB 

Molecular dynamics simulations in the presence of 

lipids on a functionally relevant fragment of myelin 

basic protein 

E. POLVERINI 1 AND G. HARAUZ 2 

1. Dept. of Physics and CNISM, University of Parma, Viale G.B. Usberti 

7/A, Parma 43124, Italy 

2. Dept. of Molecular and Cellular Biology and Biophysics 

Interdepartmental Group, University of Guelph, 50 Stone Road East, 

Guelph, Ontario, Canada N1G 2W1 

Myelin basic protein (MBP) is a multifunctional protein of the central nervous system whose 

principal role is to maintain the compactness and integrity of the myelin sheath, the multilamellar 

membrane wrapped around nerve axons that allows efficient transmission of action potentials 

along them. MBP is involved in human demyelinating diseases and it is a candidate autoantigen in 

multiple sclerosis. However, MBP also interacts with other proteins such as cytoskeletal and 

signaling proteins, adapting its structure to its different roles. The three-dimensional structure of 

MBP is still unknown, due to its intrinsic flexibility and the dependence of conformation on local 

environment. This study investigates the conformation and dynamics of a highly conserved central 

fragment of MBP, consisting of two consecutive regions with different relevant functionalities. The 

first one is associated with the membrane and comprises the primary immunodominant epitope in 

multiple sclerosis; the second one was predicted to be a ligand for SH3-domains of signaling 

proteins. Molecular dynamics simulations were performed both in aqueous environment only and 

in the presence of a dodecylphosphocholine micelle, starting from a structure extrapolated from 

experimental data [1]. The results confirm the experimental hypothesis and provide further 

information at atomic detail. In water, a flexible starting structure forms a small transient alphahelix 

in the first region, and a core of poly-proline type II (PPII) helix in the second one. In the 

micelle system, the first region remains anchored to the lipids in an alpha-helical conformation, 

while the proline-rich second region is a long PPII helix pointing outwards, ready to interact with 

signaling proteins. 

References 

[1] G. Harauz, D. S. Libich, Current Protein & Peptide Science (2009), 10(3), in press. 

84


PB 

Local flexibility of Human Serum Albumin complexed 

with fatty acids 

B. RIZZUTI 1 , M. PANTUSA 2 , R. BARTUCCI 2 , L. SPORTELLI 2 AND R. GUZZI 2 

1. Licryl laboratory CNR-INFM, University of Calabria, Ponte Bucci, Rende, I-87036, Italy 

2. Molecular Biophysics Laboratory, Dept. of Physics and CNISM Research Unit, University of 

Calabria, Ponte Bucci, Rende, I-87036, Italy 

Human Serum Albumin (HSA) is the most abundant protein in human blood plasma and provides 

the transport of a broad spectrum of molecules that include ions, drugs, hormones and fatty acids. 

Molecular dynamics has been used to investigate the structural and dynamical properties of HSA 

in solution, both in the absence and presence of palmitate molecules docked into its three highaffinity 

fatty acid binding sites. Simulations were performed under full hydration conditions for 5 

ns by using the GROMOS force-field [1]. HSA maintains its characteristic heart-shaped tertiary 

structure and almost (70%) all-alpha secondary structure. Domain III comes slightly closer to 

Domain I and deviations from the starting structures are also observed at the three interfaces of the 

protein subdomains. Each protein ensemble clusters around a single structure, as determined with 

the Jarvis-Patrick method [2], and no major domain dislocation is detected in the analysis of 

rotation vectors in the protein mainchain performed with the DynDom software [3]. Fluctuations 

in the protein backbone are more pronounced in the presence of palmitate molecules and, as a 

consequence, the protein structure equilibrates more slowly and to higher values of atomic 

deviations in the simulations. The palmitic acid molecules remain bound to HSA in their protein 

pockets because of a combination of hydrophobic and steric effects, with electrostatic interactions 

also playing an important role. In particular, the palmitate at Site 5 fluctuates around an almost 

extended conformation within the subdomain III-b with its carboxylate head attached by means of 

Coulombic interactions with both the hydroxyl group in the sidechain of Tyr401 and the 

protonated amine group of Lys525, while the first methylene groups of the lipid chain interact 

closely with the rest of the flexible Lys sidechain (see Fig. 1). The intrinsic flexibility observed in 

the simulation for the anchoring of the palmitate molecule at Site 5 could explain the higher local 

heterogeneity observed in the experiment [4] when different fatty acids are complexed with HSA 

at this site, as compared with the other two high-affinity binding site, i.e. Site 2 and Site 4. 

References 

Fig. 1 – Simulated average structure of Site 5 in HSA. 

[1] C. Oostenbrink, A. Villa, A. E. Mark, W. F. van Gunsteren, J. Comput. Chem. 25, 1656-1676 (2004). 

[2] R. A. Jarvis, E. A. Patrick, IEEE Trans. Comp. 22, 1025–1034 (1973). 

[3] S. Hayward, H. J. C. Berendsen, Proteins: Struct. Funct. Genet. 30, 144-154 (1998). 

[4] A. B. Bhattacharaya, T. Grüne, S. Curry, J. Mol. Biol. 303, 721-732 (2000). 

85


PB 

Separable influences of protein self-dynamics and lipid 

environment on H/D exchange and heat denaturing of 

membrane proteins 

H. LACZKÓ-DOBOS 1 AND B. SZALONTAI 2 

1. Institute of Plant Biology, Biological Research Centre, Hungarian 

Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary 

2. Institute of Plant Biology, Biological Research Centre, Hungarian 

Academy of Sciences, H-6726 Szeged, Temesvári krt. 62, Hungary 

To reveal the roles of lipids and proteins in membrane dynamics, wild type and desaturase deficient 

desA - /desD - mutant of the cyanobacterium Synechocystis PCC 6803 were grown at 25 °C and 

35 °C, close to its physiological low- and high-temperature limits. Thus, a large difference in lipid 

unsaturation could be generated between wild type cells since they adapt to different growth 

temperatures by altering the level of unsaturation of their poly-unsaturated lipid fatty acyl chains. In 

contrast, in the mutant cells, lipid unsaturation could not largely changed with the growth 

temperature since desA - /desD - mutant contains only mono-unsaturated fatty acyl chains. Infrared 

spectra of thylakoid and cytoplasmic membranes isolated from both types of cells were recorded in 

3 °C steps between 8 and 92 °C. By analyzing the rates of protein structural changes, hydrogendeuterium 

exchange, trans- and lateral membrane lipid disorders as functions of the temperature 

(the details of the method were described earlier [1]), we concluded that (i) in the low-temperature 

stress region, in the wild type cells, the gel-to-liquid crystalline phase transition of the lipids 

correlates with the growth temperature but does not in the desA - /desD - mutants; (ii) at physiological 

temperatures, both protein and lipid dynamics are regulating/regulated providing remarkably 

constant dynamics for the thylakoid membrane over a 15-20 °C wide temperature range; (iii) in the 

high-temperature stress region, protein structure and dynamics are changing sharply without any 

correlation with growth temperature or mutation. This finding points to the possible primacy of 

proteins as initiators of heat-shock alarms; (iv) there are substantial differences between the 

dynamics of the proteins in the thylakoid and in the cytoplasmic membranes, which can be related 

to their different biological roles. 

References 

[1] B. Szalontai, PMC Biophysics, 2:1 (2009). 

86


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Expression and biophysical characterization of exportin6 

DAVID SZATMARI, REKA DUDAS, ATTILA NAGY, GABOR HILD AND MIKLOS NYITRAI 

University of Pecs, Medical School, Department of Biophysics 

Active macromolecular transport between the nucleus and cytoplasm proceeds across nuclear pore 

complexes and is mainly regulated by the importin-β protein family. A novel family member from 

higher eukaryotes is the exportin6, which is working like a special transport receptor of the 

corresponding proteins. The structural and kinetic properties of a protein could give important 

information to understand the intramolecular events underlying its function. We are interested in 

which protein pathway proceeds the export and import of the cytoskeletal components between 

the nucleus and cytoplams? Which proteins regulate the transport from the nucleus, and how? 

There is a verified function of the exportin6, namely the profilin regulated actin export. [1] 

However, the molecular mechanisms by which the formation of the exportin6-profilin-actin 

complexes occurs are unknown. We are aiming to determine the affinities describing the 

complexes; the affinity of exportin6 epitopes for G-actin or F-actin. We also attempt to find out 

how the stability of complexes is maintained by various profilin isoforms. To achieve these aims 

we express and purify the involved proteins and carry out the biophysical characterization of 

exportin6-profilin-actin complexes. The human exportin6 (GeneBank reference ID: 

ref|NT_010393.15|Hs16_10550 Homo sapiens chromosome 16 genomic contig, reference assembly 

Length=25336229) is a 124 kDa molecular weight protein. We use a pET-19b vector that contains an 

amplicylin resistance gene. The expression of the protein was carried out in BL21 E.coli cells. The 

exportin6 was expressed with an N-terminal his-tag and purified by Ni-NTA and Co-NTA 

chromatography, in denaturant and native conditions. After the purification we obtained exportin 

6 in a few micromolar concentration. Having the protein in hand we measure the conformational 

and kinetic properties of the exportin6-profilin-actin complexes with EPR- and fluorescence 

spectroscopic, calorimetric and rapid-kinetic methods. 

References 

[1] Theis Stüven, Enno Hartmann and Dirk Görlich: Exportin 6: a novel nuclear export receptor that is specific for 

profilin-actin complexes. The EMBO Journal Vol. 22 No. 21 pp. 5928±5940, 2003 

87


PB 

Metal complexes of a plant metallothionein under 

radical stress: assessment of structural modifications 

and transfer of radical damage 

A. TORREGGIANI 1 , C. FERRERI 1 , A. TINTI 2 , S. ATRIAN 3 , M. CAPDEVILA 4 AND C. CHATGILIALOGLU 1 

1. Institute I.S.O.F. (C.N.R.) via P. Gobetti 101, I-40129 Bologna, Italy 

2. Dep. of Biochemistry, Univ. Bologna, I-40126 Bologna, Italy 

3. Dept. Genètica, Univ. Barcelona, 08028-Barcelona, Spain 

4. Dep. Quimica, Univ. Autòn., E-08193 Bellaterra, Barcellona, Spain 

Damages to Zn 2+and Cd 2+ complexes of a metallothionein (MT) from a plant (Quercus suber - Qs), 

due to radical stress exposure, were investigated. QsMT, obtained by in vivo synthesis, is a lowmolecular 

weight cysteine-rich protein with high capacity for binding metal ions. Although MTs do 

not appear to be essential for life, there is mounting evidences for a survival advantage of MT in 

situations of stress, including exposure to radicals and toxic metals. Gamma-irradiation was used 

to simulate the conditions of an endogenous radical stress. The degradation of the metal complexes 

was followed by Raman spectroscopy and the occurrence of tandem protein/lipid damage was 

shown by using a biomimetic model based on unsaturated lipid vesicle suspensions [1,2]. The H • 

and eaq – attacks on the metal-QsMT aggregates are able to induce significant structural changes 

such as partial deconstruction and/or rearrangement of the metal clusters, and breaking of the 

protein backbone. Sulfur-containing residues resulted to be selectively attacked; in particular, Cys 

resulted to be among the most sensitive residues towards radical attack, suggesting that the 

thiolate clusters of both metal-QsMTs act as efficient interceptors of reducing species (Fig. 1). 

Under reductive stress Zn-QsMT undergoes a significant thiolate group oxidation. The 

participation of His to metal coordination became necessary for protein stabilization after radical 

stress. The radical-induced effects were dependent on the divalent metal bound. The reactions of 

reductive reactive species with Met residues and/or sulfur-containing ligands afford diffusible 

sulfur-centered radicals, which migrate from the aqueous phase to the lipid bilayer and transform 

the cis double bond of the oleate moiety to the trans isomer (Fig. 1). 

References 

Fig. 1 – Raman Spectra of Zn-QsMT subjected to different gammairradiation 

doses and mechanism of the tandem radical damage. 

[1] A. Torreggiani, J. Domenech, A. Tinti, J. Raman Spectrosc. DOI 10.1002/jrs.2328 

[2] A. Torreggiani, J. Domenech, R. Oriuela, C.Ferreri, S.Atrian, M. Capdevila, C. Chatgilialoglu, Chem.Europ.J. DOI: 

10.1002/chem.200802533 

88


PB 

Temperature Dependence Radiationless Transition of 

G4 Wires and dGMP 

A. URITSKI 1 , S. KOTLYAR 2 AND D. HUPPERT 1 

1. Raymond and Beverly Sackler Faculty of Exact Science, School of 

Chemistry, Tel Aviv University, Tel Aviv 69978, Israel 

2. George S. Wise Faculty of Life Science, Department of Biochemistry, 

Tel Aviv University, Tel Aviv 69978, Israel 

Steady state and time resolved emission techniques were employed to study the nonradiative 

process of deoxygunaosine monophosphate (dGMP) and the novel uniform continuous G4 wires 

containing hundreds of stacked tetrads. We found that the time resolved emission of both dGMP 

and G4 wires decays nonexponentialy. At room temperature the short time decay of the G4 wires 

is about 10 ps. At low temperatures in ice the fluorescence quantum yield of both dGMP and G4 

wires increases as the temperature decreases. For G4 wires the fluorescence quantum yield 

increases from about 10 -3 at room temperature to about 0.03 at liquid nitrogen temperatures. The 

asymptotic long time decay of the lifetime corrected G4 wires emission obeys a power law. At all 

temperatures the average fluorescence decay time of G4 wires is longer than that of dGMP. We 

successfully used an inhomogeneous nonradiative model to fit the experimental results. 

References 

ln [k PT ] 

25.5 

25.0 

24.5 

24.0 

23.5 

23.0 

22.5 

k 1 

k 2 

22.0 

0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.010 

1/T [K -1 ] 

Fig. 1 – Arrhenius plot of ln[k PT] versus 1/T for G4 wire samples in 

ice with calculated fit. 

[1] T. Gustavssonk, A. Sharonov, M. Onidas, D. Markovitsi, Chem. Phys. Lett. 356, 49-54 (2002). 

[2] C.E. Crespo-Hernăndez, B. Cohen, M. P. Hare, B. Kohler, Chem. Rev. 104, 1977-2020 (2004). 

89


PB 

Protein motion in the ns to ms range at a molecular 

resolution 

K. WALTER 1 , N.-A. LAKOMEK 1 , M. FUNK 1 , C. FARES 1 , D. EGGER 2 , R. GULICH 2 , M. PETRI 3 , J. 

MEILER 4 , A. LOIDL 2 , M. GRUEBELE 5 , S. TECHERT 3 , P. LUNKENHEIMER 2 , S. BECKER 1 , D. LEE 1 , C. 

GRIESINGER 1 

1. Dept. of NMR based Structural Biology, Max-Planck-Institute for 

Biophysical Chemistry, Am Faßberg 11, Göttingen, D-37077, Germany 

2. Centre for Electronic Correlations and Magnetism, University of 

Augsburg, Universitätsstraße 2, D-86135 Augsburg, Germany 

3. Structural Dynamics of (Bio)chemical Systems, Max-Planck-Institute 

for Biophysical Chemistry, Am Faßberg 11, Göttingen, D-37077, 

Germany 

4. Center for Structural Biology, Vanderbilt University, 465 21st Ave 

South, Nashville, 37212, USA 

Complete geometry optimizations were performed on diverse drugs with similar side effect. The 

considered drugs were selected apart from the more common biological effects. Frequency 

calculations and geometrical optimizations were carried out at the abinitio level of theory using the 

Gaussian software. The best conformers were found for these molecules and set up apparent 

pictures. These figures are along with thermodynamic data such as thermal energies (E), thermal 

enthalpies (H) and thermal Gibbs free energies (G) have also included in this study. All of the 

mentioned drugs were revealed that the structural requirements include an electron-rich 

functional group in the molecular plane of and separated from the center of an aromatic ring. In 

order to evaluate scope and investigation of these different drugs as their similar side effect (like 

hair-loss side effect), the geometrical center of the aromatic rings were served as the most easily 

defined reference point. The electron-rich functional group was considered in the plane of and 

separated from the aromatic ring center. For the best choice of electron-rich functional group and 

also a deeper investigation in considered drugs, it was used the calculated atomic charges. Then, 

the measurement of distance parameter between the related functional group and defined 

reference point was employed Gaussian program and its correlated softwares. These kinds of 

parameters are the most important factors in explanation of relationships in structure and 

reactivity and it is used to clarify some pharmacokinetic properties and biological effects. For 

drugs with hair-loss side effect, the calculated considered distances have had high consistency 

with 95% confidence limits (ca. 7.68 angstroms). Therefore, one can recognize that the related 

distance, is important and effective factor in structure of drugs with similar side effect. Therefore, 

one can recognize that the theoretical attempts are made to circumvent to biological side effect for 

the new synthesis in experimental study. 

90


PB 

Carboxylation reactions monitored by time-resolved 

FTIR spectroscopy and caged-CO2 

G. SCHÄFER, W. MÄNTELE AND G. WILLE 

Inst. of Biophysics, Goethe-University Frankfurt, Max-von-Laue-Str. 1, 

60438 Frankfurt, Germany 

We present here a new method for the time-resolved study of carboxylation reactions using 

Fourier Transform Infrared spectroscopy (FTIR). Although compounds that release a desired 

molecule upon photoexcitation ("caged compounds") are well established, this technology has not 

been applied to the study of reactions with carbon dioxide as educt [1]. We release CO2 from 

nitrophenylacetate [2] by a laser flash and follow its subsequent reactions. Rapid-scan FTIR is 

ideally suited to this purpose as the carbon dioxide IR band is strong and set well apart from 

typical protein, buffer and water vibrations. The "caged carbon dioxide" yielded CO2 on a very fast 

time scale (~200 ps) and with sufficient quantum yield [3]. It was then employed to observe two 

exemplary reactions of carbon dioxide: the reversible formation of a carbamate with 

diethylethanolamine, and the accelerating effect of carbonic anhydrase on CO2 hydration. Both 

examples prove the applicability of the method. We then used it for the study of carbon dioxidebinding 

in Ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO). Although RuBisCO is 

the most abundant enzyme on our planet, its catalytic mechanism is still not fully understood. 

Prior to catalysis, RuBisCO must be activated by binding of carbon dioxide to a lysine in the active 

site. The resulting carbamate can then act as a ligand for a catalytically important magnesium ion 

[4]. This activation process is very difficult to study with classical methods. We could demonstrate 

that the binding of a carbon dioxide molecule in the active site is very fast, independent of the 

presence of magnesium, and that it can be inhibited with the isoelectronic azide ion. Furthermore, 

there are IR peaks consistent with the expected lysine-bound carbamate and possibly with a 

localized water molecule that is being displaced by CO2. Our new method can now be applied to 

further studies on carbon dioxide processing enzymes. In the future, we will also synthesize a 13Clabeled 

caged-CO2. This will allow the assignment of absorption bands in the fingerprint region of 

the IR spectrum to reaction intermediates and products of the enzymes. 

References 

[1] J.A. McCray, D.R. Trentham, Annu. Rev. Biophys. Biophys. Chem. 18, 239-270 (1989). 

[2] D.J .Margerum, C.T. Petrusis, J. Am. Chem. Soc. 2467-2472 (1969). 

[3] K. Neumann et al., manuscript in preparation 

[4] A.R. Portis, M.E. Salvucci, W.L. Ogren, Plant Physiol. 82, 967-971 (1986). 

91


PB 

Comparative Studies of Human Indoleamine 2,3- 

Dioxygenase and Tryptophan 2,3-Dioxygenase 

SYUN-RU YEH, ARIEL LEWIS-BALLESTER, DIPANWITA BATABYAL, TSUYOSHI 

EGAWA, CHANGYUAN LU, YU LIN 

Albert Einstein College of Medicine, Department of Physiology and 

Biophysics, 1300 Morris Park Ave, Bronx, NY 10461, USA 

In contrast to the wide spectrum of P450 monooxygenases, there are only two heme-based 

dioxygenases in humans, tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase 

(hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan 

(LTrp) to N-formyl kynurenine (NFK), the initial and rate-limiting step of the kynurenine 

pathway. Although they carry out the same reaction, the two enzymes engage in two distinct 

physiological functions: hTDO is a hepatic enzyme, which controls homeostatic serum tryptophan 

concentrations, whereas hIDO is a ubiquitous enzyme, which regulates T-cell mediated immune 

responses. Despite its importance, the mechanism by which the two enzymes execute the 

dioxygenase reaction remains elusive. We have used resonance Raman spectroscopy to study the 

structural and functional properties of hIDO and hTDO.1-3 Our data reveal the importance of 

stereoelectronic factors in hTDO and hIDO, as well as the subtle mechanistic differences between 

the two enzymes. Recently, hIDO has attracted a great deal of attention, as a result of the 

recognition of its potential as a therapeutic target for cancer. Our data offer a starting point for 

additional computational and structural investigations, which are anticipated to provide valuable 

insights for the development of anti-cancer drugs specifically targeting hIDO. 

References 

Fig. 1 – The crystallographic structure (PDB: 2NW8) of a bacterial 

analog of hTDO(a) and the hIDO and hTDO data points in the ν C-O vs 

ν Fe-CO correlation plot (b). 

[1] Batabyal D, Yeh SR., Substrate-Protein Interaction in Human Tryptophan Dioxygenase: The Critical Role of H76. J Am 

Chem Soc. 131, 3260–3270, 2009. 

[2] Batabyal D., Yeh SR., Human tryptophan dioxygenase: a comparison to indoleamine 2,3-dioxygenase. J Am Chem Soc, 

129, 15690-701, 2007. 

[3] Samelson-Jones BJ, Yeh SR., Interactions between nitric oxide and indoleamine 2,3-dioxygenase. Biochemistry. 45, 8527- 

38, 2006. 

92

Protein folding and aggregation 13 th ECSBM −−−− Book of Abstracts 

PB 

Pathogenic E4 isoform of apolipoprotein E stabilizes 

amyloid-β oligomers 

E. CERF 1 , J-M. RUYSSCHAERT 1 , E. GOORMAGHTIGH 1 , V. NARAYANASWAMI 2 AND V. RAUSSENS 1 

1. Structure and Function of Biological Membranes, Université Libre de 

Bruxelles, Boulevard du Triomphe CP206/2, Brussels, 1050, Belgium 

2. Children’s Hospital Oakland Research Institute, 5700 Martin Luther 

King Jr. Way, Oakland, California 94609, USA 

The Alzheimer’s disease (AD) is the most common neurodegenerative disorder, which provokes 

dementia in the elderly. The aggregation of the highly hydrophobic amyloid-beta peptide (Aβ) is 

most probably the early event that subsequently leads to amyloid plaques deposition in the brain, 

synapses degeneration and eventually neuronal death [1]. Although Aβ aggregation pathway still 

remains unclear, many recent studies point out the enhanced toxicity of soluble oligomers in 

comparison to monomers or insoluble fibrillar species [2]. The ε4 allele of the apolipoprotein E 

(ApoE) gene is the major known genetic risk factor in late onset AD as people carrying one ε4 

allele have significantly higher chances to develop the disease [3]. A few mechanisms underlying 

this pathogenic nature have been identified. Nevertheless, none of them have been completely 

elucidated. Our group has shown that ATR-FTIR spectroscopy could discriminate between Aβ 

oligomers and fibrils. Indeed, oligomers display anti-parallel β-sheet spectral components while 

fibrils are characterized by a parallel β-sheet organization [4]. Using those structural spectral 

features to characterize the oligomeric content of our samples, we studied here the influence of 

lipid-free ApoE on Aβ aggregation. Our experiments clearly demonstrated that the presence of 

ApoE4 prevented the formation of insoluble fibrils and decreased Aβ aggregation rate by 

stabilizing the oligomeric forms of the peptide. This result was also observed in the presence of E3 

isoform but in markedly lower proportions. Since it is now well-established that oligomers are the 

most toxic Aβ entities, the higher ability of ApoE4 to stabilize Aβ oligomers could partly explain its 

impact on AD compared to E3 isoform. 

References 

[1] J. Hardy, D.J. Selkoe, Science 297, 353-356 (2002). 

[2] M.D. Kirkitadze, G. Bitan, D.B. Teplow, J. Neurosci. Res. 69, 567-577 (2002). 

[3] E.H. Corder, A.M. Saunders, W.J. Strittmatter, D.E. Schmechel, P.C. Gaskell, G.W. Small, A.D. Roses, J.L. Haines, 

M.A. Pericak-Vance, Science 261, 921-923 (1993). 

[4] E. Cerf, R. Sarroukh, S. Tamamizu-Kato, L. Breydo, S. Derclaye, Y. Dufrêne, V. Narayanaswami, E. Goormaghtigh, 

J-M. Ruysschaert, V. Raussens, Biochem. J., accepted manuscript (2009). 

93


PB 

The mystery of reconstructive denaturation of proteins 

in the presence of certain surfactants 

M. VOLLMAR, M. DZIUK, K.-J. TIEFENBACH AND H. DURCHSCHLAG 

Institute of Biophysics and Physical Biochemistry, University of 

Regensburg, Universitaetsstrasse 31, D-93040 Regensburg, Germany 

A few proteins were reported to show a pronounced structural rearrangement of their secondary 

structure upon the impact of high concentrations of the anionic surfactant sodium dodecyl sulfate 

(SDS) [1, 2]. Pronounced reconstructive denaturation effects were found with proteins of low or 

high helix content, finally leading to a medium helicity (about 30 %); proteins of medium helix 

content adhered to their initial, intermediate helicity. Though this puzzling phenomenon is known 

since many years, details, possible generalizations, and the rationale behind the changes are 

completely unclear to date. Currently we are studying the enigmatic behavior of reconstructive 

denaturation in further detail, in particular with respect to the nature of eligible proteins 

(exhibiting different molecular characteristics such as amount of initial helicity, charge, mass, and 

number of disulfides), the surfactants used (anionic, cationic, zwitterionic, nonionic), and by 

applying different spectroscopic techniques (absorption, fluorescence, CD) under a variety of 

experimental conditions (e.g., variable concentrations of proteins and surfactants, presence of 

reducing agents). The most relevant information concerning secondary structural changes can be 

obtained from far-UV CD spectra, in combination with computer-aided approaches for evaluating 

the information obtained at several wavelengths and applying a set of reference spectra (CDNN, 

Selcon 3, Contin LL), in addition to conventional evaluations of the helix content according to 

Chen and Yang or Greenfield and Fasman at a definite wavelength (λ = 220 or 208 nm) [3]. 

Visualization procedures for protein molecules (e.g., by RasMol or PyMol) turned out to be 

indispensable tools as well, for illustrating the precise 3D structure, surface envelopes, distribution 

of amino acid (AA) residues on the protein surface and in the interior [4]. According to our results, 

the phenomenon of reconstructive denaturation is not confined to a few proteins and SDS, but 

seems to be a more general phenomenon occurring with several proteins of low or high helicity in 

the presence of anionic, cationic and zwitterionic surfactants. For example, prominent effects were 

obtained with the protein β-lactoglobulin, after the preceding impact of elevated concentrations of 

SDS, C16-trimethylammonium bromide, or C16-dimethylammoniopropane sulfonate. 

Explanations can be ascribed to the specific characteristics of the proteins and surfactants applied. 

The highly intricate balance between hydrophilicity/hydrophobicity, positive/negative charge 

and hydration on the surface and the interior of the proteins, together with the surfactant 

characteristics such as extent and nature of their hydrophilic and hydrophobic parts are 

responsible for the surfactant-induced effects. Both ionic and hydrophobic interactions seem to be 

responsible for the highly complicated interplay between proteins and surfactants, together with 

the presence/absence of certain AAs on the protein surface and the interior (e.g., presence of helixforming 

AAs, absence of too much SS bonds, presence of various positive charges and absence of 

too many negative charges on the protein surface in the case of an anionic surfactant). 

References 

[1] B. Jirgensons, Biochim. Biophys. Acta 434, 58-68 (1976). 

[2] B. Jirgensons, J. Protein Chem. 1, 71-84 (1982). 

[3] H. Durchschlag, M. Vollmar, M. Dziuk, K.-J. Tiefenbach, Jorn. Com. Esp. Deterg. 38, 213-223 (2008). 

[4] H. Durchschlag, A. Behr, M. Bartlang, M. Dziuk, D. Roderer, B. Salecker, M. Vollmar, K.-J. Tiefenbach, Jorn. Com. 

Esp. Deterg. 39, 65-76 (2009). 

94


PB 

Interaction between linker histone H1 and non-histone 

chromatin protein HMGB1 

A.V. FONIN 1, 2 , OLGA V. STEPANENKO 1 , I.M. KUZNETSOVA 1 , K.K. TUROVEROV 1 , E.I. 

KOSTYLEVA 1 , V.I. VOROBYEV 1 


av. 4, St.-Petersburg, 194064, Russia, 

2 St.-Petersburg State Politechnical University, Polytechnicheskaya, 

av.29, St.-Petersburg, 195251,Russia 

It is known that many proteins can form compact ordered structure only in contact with their 

partners, e.g. DNA or RNA molecules, different proteins, metal ions, etc. Interaction with partners 

is the main function of such intrinsically disordered proteins. Non-histone chromatin protein 

HMGB1 and linker histone H1 belong to such protein class. Both of them are the main chromatin 

proteins taking part in formation of high levels of chromatin structural organization. It can not be 

excluded that HMGB1 and H1 being the components of chromatin can interact not only with DNA 

but also with each other. There are only few works devoted to this problem and there is no 

unanimous view on it in literature. In this work the investigation of interaction between the H1 

and HMGB1 in the wide range of ionic strength solution was carried out using absorption spectra, 

intrinsic tryptophan and tyrosine fluorescence spectra, far- and near-UV CD spectra. H1 to 

HMGB1 molar ratio was varied from 1:4 to 4:1. The decrease of light scattering in HMGB1 

solutions with the increase of ionic strength suggests the destruction of associates which HMGB1 is 

known to form at low ionic strength. The increase of H1 content in solution also causes decrease of 

the light scattering, which can be explained by disintegration of HMGB1 associates due to HMGB1 

interaction with H1. So the significant decrease of HMGB1 light scattering in the presence of H1 in 

low ionic strength solutions can be regarded as the main evidence of protein interaction. The 

changes in far-UV CD spectra suggest the increase of α-helical regions in protein structure with the 

increase of H1 content in solutions of HMGB1. Intrinsic UV-fluorescence revealed only small 

changes of HMGB1 tertiary structure in the presence of H1 at all experimental conditions. 

Insignificant quenching of the HMGB1 tryptophan fluorescence was observed in all solutions 

containing both proteins. Thus, the data obtained allow us to conclude that there is interaction 

between non-histone chromatin protein HMGB1 and linker histone H1. The protein interaction is 

accompanied by the increase of ordered regions in the protein molecules and by minor changes in 

protein tertiary structure. 

95


PB 

Can insulin avoid cell degeneration induced by a-beta 

amyloid 

P. PICONE 1, 2 , R. CARROTTA 3 , D. GIACOMAZZA 3 , P.L. SAN BIAGIO AND M. DI CARLO 2 

1. Dipartimento di Chimica e Tecnologie Farmaceutiche, University of 

Palermo, Via Archirafi 32, 90123 Palermo 

2. IBIM – CNR, Via U. La Malfa 153, 90146 Palermo 

3. IBF – CNR, Via U. La Malfa 153, 90146 Palermo 

It has been recentely accepted that Alzheimer's patients also have problems with glucose 

utilization, in a way that still is not completely understood thus suggesting a relationship between 

Alzheimer’s disease (AD) and diabetes. Further some diabetes drugs appear to slow the cognitive 

decline associated with AD. It has, also, been recently demonstrated that extracellular injection of 

insulin is able to protect neurons against A-beta amyloid cell death. One of the proposed theories 

to explain such an effect is that the hematic glucose levels affect the metabolism of the 

hippocampus, a part of the brain (associated with memory, emotion and motor skills) which is 

strongly damaged in Alzheimer’s patients. The aim of this study is to investigate the effect of 

insulin on the A-beta induced degeneration and oxidative stress observed on the neuroblastoma 

LAN5 cell line. In particular, the present study looks into the role of insulin in the inhibition of Abeta 

specific degenerative apoptotic pathways. Preliminary results indicate that insulin dissolved 

in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to 

reduce neurodegeneration induced by A-beta amyloid in a dose dependent manner. The link 

between diabetes and Alzheimer's disease may provide new targets for future Alzheimer's 

treatments. Moreover, due to the increased incidence of diabetes in western countries, a deeper 

understanding of such a link is relevant in order to control the possible escalation in the number of 

people dealing with dementia. 

96


PB 

Influence of TFE on the proteins interactions of a 

lysozyme solution: a small angle X-ray scattering study. 

F. M. GIORDANO 1 , A. LONGO 1 , M. MANNO 2 , M. D’AMICO 2 , S. RACCOSTA 2, 3 AND V. 

MARTORANA 2 

1. Institute of Nanostructured Materials at Palermo, Italian National 

Research Council, Via Ugo La Malfa 153, Palermo, I-90146, Italy 

2. Institute of Biophysics at Palermo, Italian National Research Council, 

Via Ugo La Malfa 153, Palermo, I-90146, Italy 



Intermolecular proteins interactions play a fundamental role in determining the solubility, the 

misfolding and aggregation processes of proteins in solution. For instance, alcohols are known to 

affect protein interactions and conformation. Trifluoroethanol (TFE) is an alcohol, which induces 

conformational changes in proteins, either by a “macroscopic” solvent-mediated effect or by a 

direct interaction with the protein [1]. In particular for a protein model, the Bovine Serum Albumin 

(BSA), diluted at different concentration of TFE, the solvent-mediated pair wise interaction was 

investigated by small angle X-ray scattering [2]. It was found that TFE affects intermolecular 

interaction on BSA forming a preferential solvatation shell around the protein: at low TFE 

concentration it is possible to model protein-protein interaction with a repulsive hard sphere 

potential, accompanied by electrostatic repulsion. On the other hand, at TFE concentrations above 

the threshold of 16% v/v, attractive interactions become prevalent [2]. Here, we study the 

influence of the trifluoroethanol (TFE), on the intermolecular interactions of another model 

protein, the hen egg-white lysozyme, to extend the previous studies checking if the preferential 

solvatation is a general feature of protein in water-TFE mixtures. 

References 

[1] R. Walgers, T. C. Lee, A. Cammers-Goodwin, J. Am. Chem. Soc. 120, 5073-5079 (1998). 

[2] R. Carrotta, M. Manno, F. M. Giordano, A. Longo, G. Portale, V. Martorana, P. L. San Biagio, Phys. Chem. Chem 

Phys 11, 4007-4018 (2009). 

97


PB 

The thermodynamic consequences of loop mutations in 

azurin 

R. GUZZI 1 , L. SPORTELLI 1 AND C. DENNISON 2 

1. Dept. of Physics, University of Calabria, Ponte P. Bucci, Rende, I- 

87036, Italy 

2. Institute for Cell and Molecular Biosciences, Medical School, 

Newcastle University, Newcastle upon Tyne NE2 4HH, UK 

Loop-directed mutagenesis has been used to insert the amicyanin (AMI) and plastocyanin (PC) 

type 1 (T1) copper-binding loops into azurin (AZ) to obtain the chimeric variants, AZAMI and 

AZPC, respectively. In both proteins the metal binding site has typical T1 properties and the 

overall structures of AZAMI and AZPC are remarkably similar to that of AZ. The conformation of 

the loop in these chimeric proteins resembles that of the native protein. To assess the influence of 

these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been 

investigated by differential scanning calorimetry, optical absorption and fluorescence 

spectroscopy. The calorimetric profiles of both AZ variants exhibit a complex shape consisting of 

two endothermic peaks and an exothermic peak, suggesting a multistep-unfolding pathway 

similar to that observed in AMI (La Rosa et al. EBJ 2002, 30; 559-570). The thermal transition 

between the native and the denaturated states is irreversible and is scan rate dependent. The 

stability of the loop variants is noticeably reduced when compared to the wild type protein. When 

comparing the temperature of the maximum heat absorption, Tmax, the single endothermic peak of 

AZ is at 83.7 °C (at the scan rate of 60 °C/h), whereas for AZAMI and AZPC this peak is at 74.0 °C 

and 67.0 °C respectively. The temperature dependence of the absorbance at 608 nm, due to an 

active site ligand-to-metal transition, has a similar trend in AZAMI and AZPC to that observed for 

AZ, although the transition temperatures are decreased, especially for AZPC (by about 15 °C). The 

mutated cupredoxins also exhibit lower stability when investigated by fluorescence spectroscopy. 

Despite these loop mutations having limited effect on the structure of the metal binding site and 

the overall protein fold, their thermodynamic consequences are dramatic both in terms of stability 

and unfolding pathway. 

Reference 

[1] C. La Rosa, D. Milardi, D. M. Grasso, M. Ph. Verbeet, G. W. Canters, L. Sportelli, R. Guzzi, Eur. Biophys. J. 30, 

559-570 (2002). 

98


PB 

Characterizing peptide helices by site-specific coupling 

through use of isotopic labeling 

HENG CHI, AHMED LAKHANI, ANJAN ROY, TIMOTHY A. KEIDERLING 

Department of Chemistry, University of Illinois at Chicago, 

845 W. Taylor St., Chicago IL, 60607-7061 USA 

Spectroscopic determination of conformation in peptides and proteins is ultimately due to 

utilization of a physical observable dependent on coupling between repeating units in a polymeric 

chain. Determination of such couplings and their impact on the usable observables such as IR or 

Raman spectra is thus important for unraveling the spectra-structure relationships that underlie 

utilization of such techniques. In terms of vibrational spectroscopy, this is equivalent to 

determination of the off-diagonal force field components between sites in the sequence, which 

manifest themselves as frequency shifts from the isolated oscillator position (determined from 

diagonal FF). With isotopic labeling, transitions for specific sites in a peptide sequence can 

identified, and if the peptide is double labeled, couplings between the sites can be determined.[1,2] 

Since the vibrational coupling shift is small compared to the bandwidth, the relationship of 

spectral profiles obtained with various techniques, which give rise to differently weighted 

intensity patterns, can used to deconvolve the desired parameters. Here theoretical modeling of 

the expected spectral patterns is an essential part of the analysis. We have prepared a number of 

short peptides with lengths varying from 8 to 14 residues that are rich in Pro and favor a 31-helical 

geometry (poly-Pro II like). Since “random coils” are now routinely assigned as having this same 

conformation, we prepared another series based on Lys oligomers for comparison. Variants of a 

given sequence were labeled with 13C on the amide C=O in sequential and alternate positions in 

the center of the sequence, and the couplings were deconvolved using IR and vibrational circular 

dichroism (VCD) spectra of the amide I transition. In these examples Raman proved to be less 

useful in yielding differentiable intensity patterns. These couplings are small but vary in a way 

predicted by our DFT derived FF. They show a different pattern than for α-helices, previously 

published [2], and for 310-helices, currently under study. 

References 

[1] Rong Huang, Ling Wu, Dan McElheny, Petr Bour, Anjan Roy, Timothy A. Keiderling, J.Phys. Chem. B 113, 5661- 

5674 (2009); Rong Huang, Vladimir Setnicka, Marcus. A. Etienne, Joohyun Kim, Jan Kubelka, Robert P. Hammer, 

Timothy A. Keiderling, Journal of the American Chemical Society 129, 13592 -13603 (2007) 

[2] R. Huang, J. Kubelka, W. Barber-Armstrong, R. A. G. D Silva, S. M. Decatur, and T. A. Keiderling, J. Amer. Chem. 

Soc., 126, 2346-2354 (2004). 

99


PB 

Unusual structural characteristics of the 

Mycobacterium tuberculosis pentapeptide repeat protein 

MfpA 

S.KHRAPUNOV, H.CHENG, S.HEGDE, J.BLANCHARD, AND M. BRENOWITZ 

Dept. of Biochemistry, Albert Einstein College of Medicine 

1300 Morris Park Avenue, Bronx, NY 104161, USA 

Mycobacterium tuberculosis MfpA is a founding member of the Pentapeptide Repeat Protein (PRP) 

family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and 

inhibiting its activity [1]. MfpA’s size, shape and surface potential mimics duplex DNA. We have 

explored the solution structure and refolding of MfpA by fluorescence spectroscopy, circular 

dichroism (CD) and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat 

fold is described. This spectrum reveals a native structure whose β-strands and turns within the 

right-handed quadrilateral β-helix that define the PRP fold differ from canonical secondary 

structure types [2]. Differences between solution and crystallographic structures of MfpA reveal 

some intrinsic limitations of CD spectroscopy for protein secondary structure analysis. MfpA 

refolded from high temperature, urea or guanidium forms stable aggregates of monomers whose 

secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel ‘timedependent 

renaturation’ protocol yields protein with native secondary, tertiary and quaternary 

structure. The generality of ‘time-dependent renaturation’ to other proteins and denaturation 

methods is discussed. 

References 

[1] S.Hegde, M.Vetting, S.Roderick, L.Mitchenall, A.Maxwell, H.Takiff and J.Blanchard, Science 308, 1480-1483 

(2005). 

[2] S.Khrapunov, H.Cheng, S.Hegde, J.Blanchard, and M. Brenowitz, J. Biol.Chem. 283, 36290-36299 (2008). 

100


PB 

Towards an early diagnosis of Alzheimer’s disease : 

development of quantitative and conformationsensitive 

biosensor for the Aβ peptide 

E. KLEIREN 1, J-M RUYSSCHAERT 1, E. GOORMAGHTIGH 1 AND V. RAUSSENS 1 

1. Center for Structural Biology and Bioinformatics, Laboratory of 

Structure and Function of Biological Membranes, Faculté des Sciences, 

Université Libre de Bruxelles, CP 206/2, Blvd du Triomphe, B-1050 

Brussels, Belgium 

Alzheimer’s disease is the most common form of dementia worldwide and with the expansion of 

the at risk-ageing population, the tally of cases is expected to quadruple during the next decades. 

There is a widespread consensus among the medical and Alzheimer disease research communities 

that the development of an improved, sensitive, specific, non-invasive, diagnostic standard is 

mandatory. This neurodegenerative disorder reveals itself only late and, to date, reliable diagnosis 

is only indisputably obtained by the autopsy of the patient by the observation of neuropathological 

characteristics of the illness in the brain. One of these hallmarks is the presence of polymorphous 

extracellular deposits called “senile plaques”, mainly constituted of the aggregated 40-42 residue 

Amyloid-β peptide (Aβ), wich results (derives) from the proteolytic cleavage of the Amyloid 

Precursor Protein. Studies report that the Aβ peptide plays a major role in the onset of the disease 

and becomes neurotoxic upon aggregation [1]. Aβ can adopt different conformations, ranging from 

monomers to soluble oligomers and insoluble fibrils. It is now widely recognized that soluble 

amyloid oligomers are the primary pathogenic structure [1] and are correlated to the first signs of 

cognitive impairment, rather than the mature amyloid fibrils. Using attenuated total reflection - 

Fourier transform infrared (ATR-FTIR) spectroscopy, our group has recently shown that these 

species possess distinct spectral features : fibrils are characterized by a parallel β-sheet 

conformation while oligomers display an anti-parallel β-sheet structure [2]. According to these 

observations, our purpose is to develop a new reliable and highly-sensitive diagnostic tool, based 

on ATR-FTIR spectroscopy [3], that would be able to specifically detect and quantify the presence 

of the different forms of the Aβ peptide in solution. First results show that our biosensor is able to 

detect the presence of Aβ and discriminate between the oligomeric and fibrillar forms of Aβ(1-40) 

and Aβ(1-42). This conformation-sensitivity represents a major advantage compared to the already 

existing detection methods for Alzheimer’s disease. 

References 

[1] Dalghren KN, Manelli AM, Stine WB Jr, Baker LK, Krafft GA, and LaDu MJ J Biol Chem (2002) 277, 32046–32053 

[2] Cerf E, Sarroukh R, Tamamizu-Kato S, Breydo L, Derclaye S, Dufrêne Y, Narayanaswami V, Goormaghtigh E, 

Ruysschaert JM, Raussens V. Biochem J (2009) Accepted Manuscript 

[3] R. Voue M, Goormaghtigh E, Homble F, Marchand-Brynaert J, Conti J, Devouge S, De Coninck J Langmuir (2007) 

23, 949-955 

101


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Two-dimensional Raman and Raman optical activity 

correlation and factor analysis of lysozyme fibrillation 

T. PAZDERKA 1 , V. KOPECKÝ JR. 1 , K. HOFBAUEROVÁ 1,2 AND V. BAUMRUK 1 


University in Prague, Ke Karlovu 5, Prague 2, CZ-121 16, Czech Republic 

2. Institute of Microbiology, Academy of Sciences of the Czech Republic, 

Vídeňská 1083, Prague 4, CZ-142 20, Czech Republic 

Understanding of processes of amyloid fibrils formation is one of the key tasks in searching for 

proteins structural origin of human neurodegenerative diseases. Therefore, hen egg white 

lysozyme (HEWL) can serve as a good model of amyloid fibril formation. Furthermore, this 

protein is homologous to human lysozyme, which is one of the proteins that cause amyloid 

diseases [1]. Raman optical activity (ROA) and Raman spectroscopy are very powerful techniques 

for study unfolded proteins and promising experiments on lysozyme has been done [2]. Firstly, we 

model changes of ROA band shapes and positions and investigate characteristic patterns in 2D 

correlation spectra (2DCoS) [3]. Subsequently, temporal and thermal spectral changes in ROA and 

Raman spectra of HEWL (Fig. 1) were analyzed by means of factor analysis and 2DCoS. It gave us 

an opportunity to study delicate details of HEWL fibrillation and denaturation. 

Fig. 1 – A) ROA time dependency of hen egg white lysozyme (400 mg/mL, pH 3.7) 

fibrillation, B) lysozyme thermal denaturation monitored by Raman spectroscopy 

References 

[1] D. R. Booth, M. Sunde, V. Bellotti et al., Nature 385, 787–793 (1997). 

[2] E. W. Blanch, L. A. Morozova-Roche, D. A. E. Cochran et al., J. Mol. Biol. 301, 553–563 (2000). 

[3] I. Noda, Y. Ozaki, Two dimensional correlation spectroscopy: applications in vibrational and optical spectroscopy, 

Chichester: Wiley (2004). 

102


PB 

Effect of homologous oligomers in the thermal 

aggregation of a model protein 

M.R. MANGIONE, D. BULONE, V. MARTORANA AND P.L. SAN BIAGIO 

CNR - Istituto di Biofisica, Via Ugo La Malfa, 153 90146 Palermo (Italy) 

The study of protein aggregation is of great interest due to the relation between several 

pathological diseases (Alzheimer, BSE, Creutzfeldt-Jacob disease, etc.) and the formation of 

proteinaceous deposit in plaques, amyloid fibrils and filaments. The aggregation process can be 

modulated and/or governed by different factors such as pH, temperature, solvents, protein 

concentration, cosolutes, etc. In the last few years it has been demonstrated that the presence of 

proteins in different conformation or aggregation states can modify the aggregation properties of 

homologous wild type proteins. Here we present results concerning the effect of small oligomers 

(dimer and trimer) of Bovine Serum Albumin (BSA) on its thermal stability. In fact, solutions of 

commercial BSA are known to contain small amount of stable proteinaceous oligomers, probably 

formed upon the factory processing. We used HPLC technique to separate oligomers from 

monomers and to obtain clean and stable solutions of each species. In this way it was possible to 

prepare monomer solutions containing a given amounts of oligomers. By using Static and 

Dynamic Light Scattering (SDLS) to follow the thermal aggregation of so obtained systems, we 

found that the presence of oligomers slows down the aggregation process and reduces the final 

size of aggregates. 

103


PB 

Identification of free radicals induced by UV irradiation 

in collagen water solutions 

N. METREVELI 1 , L. NAMICHEISHVILI 2 , K. JARIASHVILI 2 , E. CHIKVAIDZE 2 , M. DGEBUADZE 1 , A. 

SIONKOWSKA 3 

1. Faculty of Physics and Mathematics, Ilia. Chavchavadze State 


2. Dept of Exact and Natural Sciences, Iv. Javakhishvili Tbilisi State 


3. Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87- 

100 Torun, Poland 

Electron paramagnetic resonance (EPR) method has shown that hydrogen atoms and acetic acid 

free radicals appear in surrounding acetic acid – water solution of collagen under ultraviolet (UV) 

irradiation. These free radicals interact with the collagen molecule; consequently, seven superfine 

components of EPR spectrum with the split of aH = 11.3 G and g-factor 2.001 appear. It is assumed 

that this spectrum is related to the free radical occurred on the proline residue in collagen molecule 

[1]. In order to discover •OH hydroxyl radicals even in minor concentration, spin trap 5.5dimethyl-1-pyrroline 

N-oxide (DMPO) has been applied. During the irradiation of collagen water 

solution in the presence of spin trap, EPR spectrum of the DMPO/•OH adduct has not been 

identified, while the above mentioned spectrum has been observed once the hydrogen peroxide 

H2O2 and FeSO4 were added to the sample. That means that water photolysis does not take place 

in collagen water solution due to UV irradiation. It was suggested that occurrence of hydrogen 

radical is connected with the electron transmission to the hydrogen ion. The possible source of free 

electrons can be aromatic residues, photo ionization of which takes place in collagen molecule due 

to UV irradiation [2]. 

References 

[1] N. Metreveli, L. Namicheishvili, K. Jariashvili, G. Mrevlishvili, A. Sionkowska, International Journal of Photoenergy, 

Article ID 76830, 1-4 (2006). 

[2] N. Metreveli, L. Namicheishvili, K. Jariashvili, M. Dgebuadze, E. Chikvaidze, A. Sionkowska, Journal of 

Photochemistry and Photobiology B: biology, 93(2), 61-65 (2008). 

104


PB 

Multiple aggregation pathways of GST-GFP protein 

induced by betaine and external perturbations. 

A. NATALELLO 1 , D. AMI 2 , J. LIU 3 , R. SANTARELLA 4 , A. DE MARCO 2 , AND S. M. DOGLIA 1 

1. Dept. of Biotechnology and Biosciences, University of Milano Bicocca, 

Piazza della Scienza 2, Milano, I-20126, Italy 

2.COGENTECH, Via Adamello 16, Milano, I-20139, Italy 

3. Lab. Ion Beam Eng., Chinese Academy of Sciences, Hefei 230031, 

Anhui, China 

4. Scientific Core Facilities , EMBL, Meyerhofstr. 1, Heidelberg, D-69117, 

Germany 

We investigated the multiple aggregation pathways of the model fluorescent fusion protein GST- 

GFP (glutathione S-transferase - green fluorescent protein) induced by different effectors through 

complementary techniques, as fluorescence, circular dichroism, Fourier transform infrared 

spectroscopy, dynamic light scattering, electron microscopy and biochemical methods. In 

particular, we examined the effect of the osmolyte betaine, which is expected to be a protein 

stabilizer. We found that betaine affects differently GST-GFP misfolding and aggregation 

depending on its concentration. Low betaine concentrations (5-7mM) misfold the protein, which 

precipitates in assemblies that are rich in β-sheets, inaccessible to protease, and bind thioflavine T. 

At higher betaine concentration (10-20 mM), the misfolded protein forms soluble aggregates with 

hydrodynamic radius of about 16 nm. Interestingly, at this concentration we also found that 

betaine induces the disruption of preformed aggregates into soluble assemblies. These results [1] 

demonstrate that betaine can stabilize, misfold, aggregate and disaggregate proteins depending on 

the balance between its unfavorable interaction with the protein backbone and the favorable one 

with side chains. In addition, we investigated the GST-GFP aggregation induced by thermal 

treatment, freezing and thawing cycles, pH variation, salt, DTT and H2O2 addition. We observed 

the formation of assemblies that, under certain conditions (salt, DTT , freezing and thawing cycles), 

contained the protein in a native-like structure and functional state [2]. These results indicate that 

the same protein can undergo to multiple aggregation pathways, leading to end products with 

different molecular structure and morphology. 

References 

Fig. 1 – Multiple aggregation pathway scheme of GST-GFP protein. 

[1] A. Natalello, J. Liu, D. Ami, S.M. Doglia, A. de Marco. Proteins. 75, 509-517 (2009). 

[2] A. Natalello, R. Santarella, S.M. Doglia, A. de Marco, Protein Expr. Purif. 58, 356-361 (2008). 

105


PB 

The role of metal ions in the thermal aggregation of 

Bovine Serum Albumin 

GIOVANNA NAVARRA 1 , ANNA TINTI 2 , MAURIZIO LEONE 1 , VALERIA MILITELLO 1 , ARMIDA 

TORREGGIANI 3 

1. Dipartimento di Scienze Fisiche ed Astronomiche, Università di 

Palermo and CNISM, via Archirafi 36, 90123 Palermo (Italy) 

2. Dipartimento di Biochimica, Università di Bologna, Via Belmeloro 

8/2, 40126 Bologna (Italy) 

3. Istituto I.S.O.F., Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 

40129 Bologna (Italy) 

In recent years, the study of the interaction between metal ions and proteins has provoked an 

increasing interest. One of the reasons is the involvement of metal ions in the development of some 

degenerative pathology, such as Alzheimer disease (AD), and Parkinson’s disease [1]. Several 

evidences indicate their implication in the onset of pathogenic disease: for example, the fibrils 

formation by beta amyloid peptide (i.e. the main component of the plaques) and the formation of 

soluble oligomeric aggregates, whose greater toxicity has been speculated, result to be markedly 

accelerated by zinc and copper ions presence[1, 2]. Nevertheless, has been reported that metal ions 

may act as inhibitors or promoters in aggregation processes in dependence on the metal/protein 

concentration [3, 4]. We report on thermal aggregation processes of bovine serum albumin (BSA) 

in presence of Cu(II) or Zn(II) ions. Aim of this work was to delineate the role of metal ions in the 

early stages of proteins aggregation kinetics. The growth of the aggregates was followed by 

Dynamic Light Scattering (DLS) measurements whereas the conformational changes occurring in 

the protein structure were monitored by infrared and Raman spectroscopy. Both in absence and in 

presence of metal ions, heating treatment induced a partial unfolding of the prevalently α-helix 

conformation of BSA, giving rise to a prevalent β-sheet structure. The DLS data indicated that the 

temperature of protein unfolding is not sensitively affected by the presence of Zn(II) or Cu(II) ions. 

At the contrary, oligomeric aggregates of about 20 nm was formed by native BSA and BSA-Cu(II), 

while bigger species (about 50 and 100 nm) was formed in presence of Zn(II) ions. The different 

effects played by the two types of ions were highlighted by Raman measurements. In fact, after 

heating, two different 3D coordination structures of metal-protein systems occurred. One is 

characterized by the metal coordination to the imidazole Nτ� atom of His which can promote intermolecular 

cross-linking; this manly takes place in the Zn-BSA system. The other involves metal 

binding to the Nτ�/ Nπ�-histidinate anion as well as to main-chain amide nitrogens, probably leading 

to an intra-molecular chelation in the Cu-BSA system. This different metal coordination also 

induced differences in local disulfide conformation and in Tyr environment. 

References 

[1] A.I. Bush, W.H. Pettingell, G. Multhaup, M. Paradis, et al., Science 265, 1464-1467 (1994) 

[2] K. Garai, P. Sengupta, B. Sahoo, S. Maiti, Biochem. Biophys. Res. Commun. 354, 210-215 (2006) 

[3] M.A. Lovell, C. Xie, W.R. Markesbery, Brian Res. 823, 88-95 (1999) 

[4] G. Navarra, M. Leone, V. Militello, Biophys. Chem. 161, 52-61 (2007) 

106


PB 

The influence of salt substitutes on the secondary 

structure of food proteins – a vibrational spectroscopic 

study 

N. PERISIC 1 , N. K. AFSETH 1, R. OFSTAD 1 AND A. KOHLER 1,2,3 

1 Centre for Biospectroscopy and Data Modelling, Matforsk/Nofima 

Food, Ås, Norway 

2 CIGENE – Center for Integrative Genetics, University of Life Sciences, 

1432 Ås, Norway 

3 Department of Mathematical Sciences and Technology (IMT), 

Norwegian University of Life Sciences, Ås, Norway 

Salt (NaCl) is the world’s most established food additive because of its beneficial effects, such as 

prolonged preservation, better sensorial properties and increased food processability. Although 

sodium is required for normal human body functions, the actual intake in a majority of <strong>European</strong> 

countries is three times higher than necessary (8-12 g/day) [1,2]. Several epidemiological studies 

have demonstrated that high sodium intake is associated with an increased risk of high blood 

pressure, which is a significant factor for cardiovascular disease and strokes development [3,4]. 

Food producers are thus working to reduce the content of NaCl and find feasible salt substitutes 

for use in food products. Sodium chloride affects a number of functional properties in meat and 

fish products, ranging from hydration and water binding capacities to textural properties. While 

new products are developed, where NaCl contents are reduced and substituted with other salts, 

there is a need to increase the understanding of how these changes affect protein structure, water 

holding capacities and textural properties of the new products. Vibrational spectroscopy 

techniques are feasible tools for studying protein structural changes, and previous studies have 

shown that the Amide I band in FTIR spectra (1700 – 1600 cm -1) is highly sensitive to the changes 

in the secondary structure of proteins. Thus, in the present study, vibrational spectroscopic 

techniques like FT-IR and Raman spectroscopy are used to monitor the structural changes in 

proteins due to the addition of different salt substitutes. Two model systems have been established 

to investigate how salt substitutes like MgSO4 and KCl affects protein structure: (1) aqueous 

solutions of pure protein, and (2) low-lipid bovine meat. In the first step, the protein solution is 

used to determine the structural changes of globular proteins that have occurred in the interaction 

with salt ions. Subsequently, this is to be used for further explanation of the structural changes in 

far more complex matrices, such as bovine meat samples. 

References 

[1] Brandsma, I., 2006, Reducing sodium, a <strong>European</strong> perspective, Food Technology 03.06 pp 24-29. 

[2] Kilcast, D. and Angus, F., 2007, Reducing salt in foods, practical strategies. CRC Press, Cambridge; England 

[3] Intersalt. 1988. An international study of electrolyte excretion and blood pressure. Intersalt Cooperative Research 

Group. Brit. Med. J., 279:319-328. 

[4] Cook, N.R. et al. (2007) Long term effects of dietary sodium reduction on cardiovascular disease outcomes: 

observational follow-ups of the trials of hypertension prevention. British Medical Journal, April 28, 334 (7599):855. 

107


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Structural organization of DNA-protein complexes of 

chromatin studied by vibrational and electronic circular 

dichroism and atomic force microscopy 

A. POLYANICHKO 1 AND H. WIESER 2 

1. Dept. of Molecular Biophysics, Faculty of Physics of Saint-Petersburg 

State University, 1 Uljanovskaya Str., Stary Petergoff, Saint-Petersburg, 

198504, Russian Federation 

2. Dept. of Chemistry, University of Calgary, 2500 University Dr., 

Calgary, Alberta, T2N 1N4, Canada 

Structure and functioning of chromatin is determined by interactions of DNA with numerous 

nuclear proteins. The most abundant and yet not completely understood non-histone 

chromosomal proteins are those belonging to a High Mobility Group (HMG) superfamily [1]. The 

object of our study is a member of the family named HMGB1. The characteristic feature of this 

protein is HMGB DNA-binding domain demonstrating rather unusual structural and functional 

properties [2]. The interplay of this protein on DNA with linker histone H1 and other proteins 

determines both structure and functioning of the chromatin. A combination of UV and IR 

absorption and circular dichroism (CD) spectroscopy was applied to investigate the structure and 

formation of large supramolecular DNA-protein complexes. This combination of techniques was 

used to overcome limitations of UV-CD (ECD) spectroscopy due to considerable light scattering in 

such solutions [3]. Based on the analysis of FTIR and UV circular dichroism spectra the interaction 

of DNA with high-mobility group non-histone chromatin protein HMGB1 and linker histone H1 

was studied. We have also studied the influence of biologically relevant metal ions, such as Mn2+ 

and Ca2+, on the structure of the DNA-protein complexes. The data obtained showed that under 

the conditions of the experiment (15 mM NaCl, protein/DNA ratio r < 1 w/w) the proteins did not 

reveal any AT or GC specificity in binding to DNA. In the presence of both proteins, mainly 

interactions in the DNA minor groove were observed, which were attributed to HMGB1 binding. 

Histone H1 facilitated binding of HMGB1 to DNA by interacting with the sugar-phosphate 

backbone and binding of Asp and Glu amino acid residues of HMGB1. Acting together, HMGB1 

and H1 stimulated the assemblage of supramolecular DNA-protein structures. The structural 

organization of the ternary complexes is modulated by the interactions between HMGB1 and H1 

molecules. The DNA-proteins interactions in the presence of metal ions were different, causing 

prominent DNA compaction and formation of large inter-molecular complexes. The interactions of 

Mn2+ take place now mostly in the major groove of DNA involving N7(G), while interactions 

between Mn2+ and DNA phosphate groups are weakened by histone molecules. Considerable 

interactions of Mn2+ ions with Asp and Glu amino acid residues of the proteins were also 

detected, while Ca2+ interacts directly to HMGB DNA binding domain. 

References 

[1] Johns E.W. The HMG chromosomal proteins, Academic Press Inc., London (1982). 

[2] Bustin M., Reeves R. Prog. Nucleic Acid Res. Mol. Biol., 54, 35-100 (1996). 

[3] Polyanichko A, Wieser H. Biopolymers, 78, 329-339 (2005). 

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FTIR and CD spectroscopy investigations of the 

structure and function of an intrinsically disordered 

plant desiccation stress protein (LEA7) in the hydrated 

and dry state 

A.V. POPOVA 1 , M. HUNDERTMARK 1,2 , R. SECKLER 3 AND D. K. HINCHA 1 

1. Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 

1, D-14476 Potsdam, Germany 

2. present address: UMR 1191 Physiologie Moleculaire des Semences, 16 

Bvd Lavoisier, 49045 Anders Cedex 1, France 

3. Institute for Physical Biochemistry, University of Potsdam, Karl- 

Liebknecht-Str. 24-25, D-14476 Potsdam, Germany 

Late Embryogenesis Abundant (LEA) proteins have been first discovered in plant seeds and were 

later also found in vegetative organs of plants, and in some bacteria and invertebrates under 

environmental stress conditions such as cold, salinity, or desiccation [1]. LEA proteins are 

generally assumed to play an important role in cellular desiccation tolerance, but their molecular 

functions are not well understood. Some have been shown to stabilize desiccation sensitive 

enzymes, to prevent dehydration-induced protein aggregation or to stabilize biological 

membranes against desiccation injury. LEA proteins are very diverse but share some common 

features such as small size and high hydrophilicity, and most are predicted to be natively 

disordered under fully hydrated conditions [2]. On dehydration it has been shown that some LEA 

proteins gain secondary structure. Here, a structural and functional characterization of the protein 

LEA7 from Arabidopsis thaliana is presented. CD spectroscopy showed that under hydrated 

conditions 80% of the protein is unstructured, but that after drying this is decreased to about 30% 

with the α-helical content increased. LEA7 was able to preserve the activity of the sensitive 

enzyme lactate dehydrogenase (LDH) during drying and FTIR analysis showed that the structure 

of both LEA7 and LDH were mutually influenced. The massive desiccation-induced aggregation of 

proteins in leaf extracts from Arabidopsis thaliana was prevented by the presence of LEA7. Under 

fully hydrated conditions the structure of LEA7 was not influenced by the presence of model 

membranes, while in the dry state LEA7 interacted with model membranes. This interaction had 

clear effects on the structure of both membranes and protein, as revealed by FTIR spectroscopy. 

References 

[1] J. Ingram, D. Bartels, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377-403 (1996). 

[2] M. Hundertmark, D. K. Hincha, BMC Genomics 9, 118 (2008). 

109


PB 

Unusual stabilizing properties of trehalose/maltose 

binding protein from Thermococcus litoralis make it a 

good candidate as a sensitive element in biosensor 

systems for sugar control 

O.I. POVAROVA 1 , OLGA V. STEPANENKO 1 , A.I. SULATSKAYA 1 , I.M. KUZNETSOVA 1 , K.K. 

TUROVEROV 1 , M. STAIANO 2 , A. VITALE 2 , S.D'AURIA 2 

1. Institute of Cytology of the Russian Academy of Sciences, 

Tikhoretsky av. 4, Saint-Petersburg, 194064, Russia 

2. Institute of Protein Biochemistry CNR, Via Pietro Castellino 111, 

Napoli, 80131, Italy 

It is difficult to overestimate the possibility to perform continuous, non-invasive monitoring of the 

glucose level in blood of diabetic patients. The potential use of glucose-binding protein (GBP) as a 

probe for the development of advanced biosensors for monitoring the level of glucose in the blood 

of diabetic patients has been already shown. Nonetheless, investigations in this direction meet 

several problems. Among them are relatively low stability of GBP and too high glucose binding 

constant. Both problems can be solved if we use trehalose/maltose binding protein from T. 

litoralis. Firstly, as D-glucose is not natural ligand of TMBP its sufficiently high dissociation 

constant (3–8 mM) corresponds to the content of glucose in human blood in normal (3.9 – 5.9 mM). 

Secondly, the stability of TMBP is in several times higher than GBP from mesophilic organism 

(E.coli). The stability of TMBP and its complex with D-glucose (TMBP/Glc) was studied by 

intrinsic fluorescence and circular dichroism in far-UV region. All recorded characteristics remain 

stable up to 3.8 M of GdnHCl and dramatically change in the narrow region of GdnHCl 

concentration (3.8–4.2M GdnHCl). TMBP being a hyperthermophilic protein remains native up to 

95 °C. The melting temperatures for TMBP and TMBP/Glc were reached only for proteins in 2.8 M 

GdnHCl. On the basis of protein denaturing by GdnHCl at different temperature the difference of 

free energy in native and unfolded state were determined for different temperatures both for 

TMBP and TMBP/Glc. 

Fl. intensity 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

I 320 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

0.2 0.4 0.6 0.8 1.0 1.2 

I 365 

0 1 2 3 4 5 6 

[GdnHCl], M 

0 

-2 

-4 

-6 

-8 

-10 

Fl. intensity 

[θ222]x10 -3 , deg cm 2 dmol -1 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

110 

ΔG0, kcal/mol 

6 

4 

2 

0 

-2 

-4 

T, 0 -6 

0 20 40 60 80 100 

C 

1.3 

1.2 

1.1 

1.0 

0.9 

0.8 

0.7 

0.6 

Temperature, 0 0 20 40 60 80 

0.5 

100 

C 

Parameter A


PB 

Fibrillogenesis of Hen Egg-White Lysozyme at acidic pH 

S. RACCOSTA 1,2 , V. MARTORANA 1 , M. MANNO 1 AND P. L. SAN BIAGIO 1 

1. Inst. of Biophysics (O.U. Palermo), Italian National Researh Council, 

Via U. La Malfa 153, Palermo, I-90146, Italy 



The formation of amyloid fibrils has a relevant interest in medical research, since several 

neurodegenerative pathologies (Parkinson’s, Alzheimer’s and Creutzfeldt-Jakob diseases, cystic 

fibrosis, diabetes Type II, etc) are linked to these highly structured protein deposits in living 

tissues. Several proteins, also not related to pathologies, can form fibrils under given 

environmental or thermodynamic conditions that affect their native conformation. A basic 

comprehension of the molecular mechanism of amyloid formation and growth is fundamental in 

order to control and inhibit the critical step of this harmful process. We use Hen Egg White 

Lysozyme (HEWL) as a model system to study the self-assembly of amyloid fibrils at acid pH. 

Indeed, the best known method to produce fibrils of wild-type lysozyme consists of keeping a 20 

mg/ml pH 2 protein solution at 65 °C for a few days [2,3]. Also, aggregation studies at acidic pH 

are of particular interest, since they elicit the role of repulsive electrostatic interaction in protein 

unfolding and self-assembly. We performed Differential Scanning Calorimetry (DSC) experiments 

of lysozyme solutions at pH 2. These measurements confirm that lysozyme is mainly unfolded 

above 60 °C, consistent with previous works. Also, we monitored the kinetics of aggregation by 

Photon Correlation Spectroscopy (PCS) for some days, in order to study the growth of aggregate 

size and their structure. The physical parameters used to understand the mechanism of the 

aggregation process were protein concentration and temperature. Our results show that the 

fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with 

quadratic dependence upon time. This behaviour recalls a simple kinetic model (proposed by 

Oosawa and Asakura) of nucleation and elongation, with nuclei in equilibrium with monomers. At 

the end of the incubation at high temperature, we observed diluted samples by Atomic Force 

Microscopy (AFM). The AFM images show linear fibrils with a diameter of a few tens of 

nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. 

Interestingly, the fibrils morphology exhibits no relevant secondary mechanisms like branching or 

thickening[1]. Indeed, this is consistent with the non-exponential growth observed in light 

scattering experiments. In order to elicit the role of repulsive electrostatic interaction in protein 

unfolding and self-assembly we extended our study at different pH. 

References 

[1] C. B. Andersen, H. Yagi, M. Manno, V. Martorana, T. Ban, G. Christiansen, D. E. Otzen, Y. Goto, C. Rischel, 

Biophys. J. 96, 1529-1536 (2009). 

[2] L. N. Arnaudov, R. de Vries, Biophys. J. 88, 515-526 (2005). 

[3] M. R. H. Krebs, D. K. Wilkins, E. W. Chung, M. C. Pitkeathly, A. K. Chamberlain, J. Zurdo, C. V. Robinson, C. M. 

Dobson, J. Mol. Biol. 300, 541-549 (2000). 

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Self aggregation mechanisms in fungal proteins by 

AFM studies 

F. SBRANA 1 , D. FANELLI 2 , L. PAZZAGLI 3 , G. CAPPUGI 3 , M.VASSALLI 4 AND B. TIRIBILLI 5 

1. Dept. of Physics, University of Firenze, Via Sansone 1, Sesto 

Fiorentino, Firenze, I-50019, Italy 

2. Dept of Energetics, University, of Firenze, Via S. Marta 3, Firenze I - 

50139, Italy 

3. Dept. of Bioch. Science, University of Firenze, Viale Morgagni 50, 

Firenze I-50134 – Italy 

4. Biophysics Institute (IBF – CNR), Via De Marini 6, Genova I-16149 

Italy 

5. Inst. for Complex Systems ISC – CNR – Via Madonna del Piano 10, 

Sesto Fiorentino, Firenze I-50019, Italy 

We report on AFM studies concerning two fungal secreted proteins, Cerato-ulmin (CU) and Cerato 

platanin (CP), in which the morphological analysis of in vitro aggregates has pointed out some 

simple self aggregation mechanisms. Cerato-ulmin is a fungal toxin class II hydrophobin involved 

in Dutch elm disease. The formation of hydrophobin films at the air-water interface plays an 

important role at different stages of the fungal development. Protein aggregation of CU has been 

obtained in vitro and samples were imaged with AFM on mica substrates. Images reveal that the 

system self-organizes in almost one dimensional pearl necklace-like chains, which subsequently 

collapse and possibly merge to form extended and rather compact planar films. We propose and 

verify a simple geometrical argument to describe the self aggregation mechanism in terms of a 

progressive thickening of the pearl chains due to the successive merging and collapse of the 

elementary constitutive units. Cerato-platanin, the first member of the “Cerato-platanin 

family’’[1,2], is a moderately hydrophobic protein produced by Ceratocystis fimbriata, the causal 

agent of a severe plant disease called Canker stain. The protein is localized in the cell wall of the 

fungus and it seems to be involved in the host-plant interaction inducing both cell necrosis and 

phytoalexin synthesis. The AFM characterization of CP aggregates deposited on mica substrates 

reveal the formation of early annular-shaped oligomers that tend to form large macrofibrillar 

assemblies. The annular-shaped oligomers seem to be the fundamental bricks of a hierarchical 

aggregation process. A simple model, based on the hypothesis that the aggregation is energetically 

favourable when the exposed CP aggregate surface is reduced, is suggested and substantiated with 

the measured aggregates’ shape and size. The proposed models can contribute to shed new light 

onto the crucial mechanism by which CU, CP and many other fungal surface proteins exert their 

effects, e.g. interact with their hosts. 

References 

[1] Pazzagli L, et al. J Biol Chem 274:24959–24964 (1999). 

[2] F. Sbrana et al. <strong>European</strong> Biophisical Journal, Vol 36 pag.727-732 (2007). 

112


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Are the properties of milk proteins influenced by 

perfluorinated contaminants? 

C. SCHWIEGER, M. AUDEBRAND AND M. H. ROPERS 

Institut Nationale de la Recherche Agronomque (INRA) Nantes-Angers 

Unité Biopolymers, Interfaces, Assemblies (BIA) 44316 Nantes, France 

During the last years it was found that perfluorinated surfactants, originating from industrial 

processes (by-, wast- and degradation products), contaminate nature. Due to their high stability 

they bioaccumulate and enter the food chain. Perfluorooctanoic acid (PFOA) is one of the final 

products of degradation of numerous fluorochemicals. It was found different body tissue, in blood 

and in cow milk. However the impact on health and food quality is not yet clear. Therefore we 

investigated the binding of PFOA to whey proteins (β-lactoglobilin, α−lactalbimin and bovine 

serum albumin) in order to elucidate the interaction mechanism as well as the impact of PFOA on 

the structure and the thermal stability of the proteins. The interactions in aqueous bulk were 

thoroughly studied by isothermal titration and differential scanning calorimetry, by infrared and 

fluorescence spectroscopy. Since whey proteins are widely used as in food industry to stabilize 

foams and emulsions, the effect of the contaminants was furthermore studied at the air-water 

interface, by means of infrared reflection absorption spectroscopy (IRRAS). We will show that that 

PFOA interacts only weakly with b-lactoglobulin and α-lactalbumin, whereas it is strongly 

interacting with bovine serum albumin. However, PFOA influences the thermal unfolding of all 

three whey proteins and it strongly influences its interfacial behaviour. 

113


PB 

Spectral properties and factors determining high 

quantum yield of thioflavin T incorporated in amyloid 

fibrils 

A.I. SULATSKAYA 1, 2, K.K. TUROVEROV 1, I.M. KUZNETSOVA 1 


av. 4, St.Petersburg, 194064, Russia, 

2 St.Petersburg State Polytechnical University, 29 Polytechnicheskaya st., 

St.Petersburg, 195251,Russia 

Thioflavin T (ThT) is widely used for studying amyloid fibrils structure and formation . 

Nevertheless, till now there is no common opinion concerning the reasons of the increase of 

fluorescence quantum yield of ThT incorporated in amyloid fibrils. Absorption spectrum, 

fluorescence lifetime and fluorescence intensity of ThT recorded in a wide range of dye 

concentration allowed to show the failure of the concepts according to which ThT incorporates in 

amyloid fibrils, in the form of dimers, eximers, or micells. All experimental data prove the model 

according to which ThT incorporates in amyloid fibrils in monomer form. The binding occures in 

“channels” that run along the long axis of amyloid fibril . Fluorescence quantum yield of ThT 

recorded in a wide range of solvent temperatures and viscosity confirmed the assumption that the 

increase of quantum yield of ThT incorporated in amyloid fibrils is caused by restriction of 

benzthiazole and aminobenzene rings torsion fluctuations about each other. The presence of the 

methyl group at nitrogen atom in benzthiazole ring makes nonplanar conformation of ThT 

energetically profitable and decreases barrier of intramolecular rotation of benzthiazole and 

aminobenzole rings relative to each other. Therefore in water solutions ThT represents dynamic 

ensemble of molecules with π-conjugated bonds of the benzthiazole and aminobenzene rings 

disturbed in various degree and the part of molecules would have the angle between the planes of 

the rings close to 90 ˚. In this case the isolated fragments of ThT should have shorter wavelength 

positions of the absorption and fluorescence spectra in comparison with those of the whole ThT 

molecule. It explains the existence of short wavelength bands of ThT fluorescence and fluorescence 

excitation spectra in solvents with low viscosity, e.g. in aqueous solutions at a room temperature. 

ThT absorption spectrum in the water solution of amyloid fibrils represents superposition of the 

absorption spectrum of free dye molecule and that incorporated in the amyloid fibrils. It was 

determined that ThT incorporation in amyloid fibrils is accompanied by strong long-wavelenght 

shift of the absorption spectrum (from 413 to 450 nm) and increase in molar extinction coefficient 

(from 26620 to 34000 M -1.cm -1 and 45000 M -1.cm -1 for insulin and lysozyme fibrills, respectively) in 

the spectrum maximum. The short-wavelength position of ThT absorption spectrum in water 

solution is explained by the fact that the ground state is stabilized by the orientational interactions 

of the polar solvent dipoles with the positively charged ThT fragments, whereas the configuration 

of the solvation shell of the ThT molecule in the excited Franck-Condon state is likely far from 

being equilibrium. The increase of molar extinction coefficient of ThT on incorporation in amyloid 

fibrils can be explained by the increase of the conjugation of π-electron system of the benzthiazole 

and aminobenzene rings because at incorporated molecules are more plane. 

114


PB 

Competition between folding de novo and aggregation 

of enhanced green fluorescent protein (EGFP) – 

spectroscopic studies 

J. KRASOWSKA 1, M. OLASEK 1, A. BZOWSKA 1, P. L. CLARK 2 AND B. WIELGUS-KUTROWSKA 1 

1. Dept. of Biophysics, Institute of Experimental Physics, University of 

Warsaw, świrki iWigury 93, Warsaw, 02-089, Poland 

2. Dept. of Chemistry and Biochemistry, University of Notre Dame, 251 

Nieuwland Science Hall, Notre Dame, IN 4656-5670 USA 

GFP is a small (238 aa), globular protein with a β-barrel structure and a central α-helix, isolated 

from the Pacific jellyfish, Aequoria Victoria [1]. In correct native structure three residues in the 

central helix – S65, Y66 and G67 – cyclize to form a chromophore able to emit visible light with a 

maximum of emission at 508 nm. Its absorbance spectrum has a complicated shape consisting of 

two peaks (395 nm and 470 nm) arising from two ionic forms of the chromophore: neutral phenol 

and anionic phenolate [2]. EGFP (enhanced Green Fluorescent Protein, mutant S65T/F64L - GFP) 

has maximum of excitation at 489 nm and maximum of emission at 509-511 nm (quantum yield 

0.6) [2]. The S65T mutation simplifies the absorption spectrum of GFP and the F64L mutation 

enhances the fluorescence. Due to its unusual chromophore GFP has became a widely used 

reporter protein in molecular biology and biotechnology. It can be easily fused to any protein of 

interest and coexpressed in cells; the GFP fluorescence is then used to visualize the distribution, 

transport and aggregation of the protein in the cell [3] (although GFP has a tendency to aggregate 

itself). The importance of GFP has been appreciated and the scientists who discovered and studied 

it were awarded the Nobel Prize in Chemistry in 2008. Recently, much interest has arisen around 

the competition between proper folding and aggregation of proteins due to the role of these 

processes in various human neurodegenerative diseases. Moreover, aggregation causes problems 

in the production and purification of large amounts of proteins during their overexpression in 

bacterial or yeast cells. Because of those the promotion of productive protein folding, and the 

suppression of competing, off pathway aggregation processes, are in major medicinal, biochemical 

and biotechnological interest. Here, in contrast to other studies describing unfolding and refolding 

processes [4,5], we have undertaken spectroscopic experiments of EGFP folding de novo and 

aggregation, in order to further develop our understanding of this important biotechnological and 

cell biological tool. We have performed equilibrium and kinetic studies by monitoring changes of 

tryptophan and chromophore fluorescence emission, changes of chromophore absorption, and also 

light scattering at 640 nm, as a function of guanidinium hydrochloride concentration and as a 

change of pH. A range of reducing agents has been also checked in order to identify conditions 

under which the efficiency of EGFP folding de novo is highest. 

References 

[1] O. Shimomura, F. H. Johnson and Y. Saiga, J. Cell. Comp. Physiol., 59: 223-229 (1962) 

[2] R. Y. Tsien, Annu. Rev.Biochem. 67, 509-44 (1998) 

[3] “Green Fluorescent Protein – Properties and Applications” edited by Chalfie M and Kain S, New York: Willey-Liss 

Inc. (1998) 

[4] S. Enoki, K. Saeki, K. Maki and K. Kuwajima, Biochem. 43, 14238-14248 (2004) 

[5] J. Huang, T. D. Craggs, J. Christodoulou and S. E. Jackson, J. Mol. Biol., 370, 356-371 (2007) 

115

Single molecule and single cell spectroscopy 13 th ECSBM −−−− Book of Abstracts 

PB 

Raman spectroscopic investigations on breast cancer 

cell lines 

THOMAS BOCKLITZ 1 , MELANIE PUTSCHE 1 , CARSTEN STÜBER 3 , JOSEF KÄS 3 , PETRA RÖSCH 1 AND 

JÜRGEN POPP 1,2 


Helmholtzweg 4, D-07743 Jena, Germany 

2. Institute of Photonic Technology, Albert-Einstein-Strasse 9, 

D-07745 Jena, Germany 

3. Institute of Soft Matter Physics, University of Leipzig, Linnéstr. 5, 

D-04103 Leipzig, Germany 

Cancer is a fatal disease, 22.4 million people lived with the disease and 6.2 million died of 

malignant neoplasia (2000). This represents an increase of 18% mortality since 1990 [1]. The 

worldwide cancer mortality-to-incidence ratio is nearly 40%, but the 5-year relative survival rates 

are strongly dependent on the stage and grade of the tumor when it is diagnosed. For breast cancer 

this effect is obvious if the 5-year relative survival rates of the local stage of tumor (97.5%) is 

compared with the distant stage (25.5%) [1]. Thus, cancer must be diagnosed and treated in an 

early stage. Since Raman spectroscopy monitors the chemical composition of a sample it can be 

used to investigate the chemical changes introduced by a disease [2], [3]. In order to display the 

ability of Raman spectroscopy as clinical diagnosis tool for breast cancer, epithelial cell lines were 

investigated. It was shown that supervised pattern recognition techniques like Linear Discriminant 

Analysis (LDA), Artificial Neural Network (ANN) and Support Vector Machine (SVM) are capable 

to classify malignant and benign cells. The identification rate which was estimated using a 

Holdout method was 99:11% while the sensitivity and the specificity were 99:71% and 96:62%, 

respectively. In order to achieve such properties it was necessary to focus on one compartment of 

the cell. The cell nucleus was chosen because the nucleus of malignant cells show major differences 

compared with a benign cell nucleus. This property is also used from pathologists for cancer 

diagnosis by visual inspection. 

References 

[1] J. Helfmann, U. Bindig, B. Meckelein, K. Wehry, N. Röckendorf, D. Schädel, M.A. Schmidt, M. Bürger, and A. Frey, 

“Early Diagnosis of Cancer (PLOMS)”, in Biophotonics: Visions for Better Health Care, edited by J. Popp and M. 

Strehle, Wiley-VCH, 231 (2006) 

[2] M. Harz, R. A. Claus, C. L. Bockmeyer, M. Baum, P. Rösch, K. Kentouche, H.-P. Deigner, and J. Popp, Biopolymers, 

8, 317-324 (2006). 

[3] M. Harz, M. Kiehntopf, S. Stöckel, P. Rösch, T. Deufel, and J. Popp, Analyst, 133, 1416-1423 (2008). 

116


PB 

TERS on bio membrane related structures 

R. BÖHME 1 , M. RICHTER 1 , P. RÖSCH 1 , V. DECKERT 1,2 AND J. POPP 1,2 



2. Institute of Photonic Technology (IPHT), Albert-Einstein-Straße 9, 


To understand biological processes located on cellular surfaces, an explicit description of the 

underlying molecular mechanisms is required. Although conventional Raman spectroscopy allows 

the investigation of even small structural and chemical modifications, it cannot achieve spatial 

resolution on the nanometer scale, where many crucial processes are expected to take place. 

Combining Atomic Force Microscopy (AFM) with Surface Enhanced Raman Spectroscopy (SERS) 

results in so called Tip-enhanced Raman spectroscopy (TERS). [1] In this case the spectral 

information stems from a very small scattering volume, hence, a spatial resolution down to 10nm 

and below is possible. [2] Motivated by investigations on biological structures like single RNA 

strands [3], virus particles [4] and bacteria [5], we aim towards the description of cellular surfaces 

on a nanometer scale. In addition to direct TERS measurements on eukaryotic cells, we focus on 

supported lipid bilayers (SLB) labeled with proteins acting as models for bio membranes. TERS 

spectra, recorded on these SLB structures, present a way to investigate the local distribution of 

protein and lipid domains. Accordingly, either Raman spectra characteristic for lipid or protein 

contribution can be found and correlated with the synchronously determined AFM images. This, 

combination of topographic and spectral information consequently provides direct bio molecular 

imaging with nanometer scaled resolution without the need for any labeling. Hence, this approach 

provides an initial step towards a label free and fast description of molecular processes located 

into a bio membrane. 

References 

[1] R. Stöckle, Y. Suh, V. Deckert, R. Zenobi, Chem Phys Lett 318, 131-136 (2000) . 

[2] A. Downes, D. Salter, A. Elfick, J. Phys. Chem. B 110, 6692-6698 (2006) 

[3] E. Bailo, V. Deckert, Angew. Chem., Int. Ed. 47, 1658-1661 (2008). 

[4] D. Cialla, T. Deckert-Gaudig, C. Budich, M. Laue, R. Möller, D. Naumann, V. Deckert, J. Popp, J. Raman Spectrosc. 

40, 240-243 (2009). 

[5] U. Neugebauer, P. Rösch, M. Schmitt, J. Popp, C. Julien, A. Rasmussen, C. Budich, V. Deckert, ChemPhysChem 7, 

1428-1430 (2006). 

117


PB 

Inorganic Probes of Protein & Cellular Environments 

L. COSGRAVE 1 , M. DEVOCELLE 2 , N. MORAN 2 , R.FORSTER 1 AND T.E. KEYES 1 

1. School of Chemical Sciences, Dublin City University, Glasnevin, 


2. Royal Society of Chemistry, St. Stephens Green West, Dublin 2, 

Ireland. 

Ruthenium polypyridyl complexes exhibit a range of photophysical properties that make them 

ideally suited as probes for the life sciences. They are photostable, absorb and emit light in the 

visible spectral region, display a large Stokes shifted and polarised emission and have long 

luminescent lifetimes rendering them environmentally sensitive. Muted for a long time as 

potential regents for cell imaging, they have not heretofore been applied significantly in this area 

because they are incapable of crossing the cell membrane. In this contribution we describe our 

approach to overcome this problem: whereby we conjugated ruthenium polypyridyl complexes to 

a carrier peptide; octoarginine (Arg8). Octoarginine is a cell penetrating peptide that enters the cell 

via endocytosis and transports the inorganic dye with it. There is no requirement for cell damaging 

permeablisation protocols or for fixing the cells, so live cells can now be imaged with these 

reagents. Importantly, using these materials cell imaging can be achieved via both resonance 

raman and luminescence imaging techniques due to the large Stokes shift of the ruthenium 

molecules. This reduces interference in the resonance raman method and increases the information 

available from an imaged cell. In the second part of this contribution we describe the use of 

ruthenium probes in direct labelling of the platelet glycoprotein GPIIbIIIa via the carbohydrate 

conjugation. GPIIbIIIa is transmembrane protein of the blood platelet, central in thrombosis and 

haemostasis, which undergoes conformational change and clustering at the platelet surface which 

renders it high affinity for its primary ligand fibrinogen as clot formation proceeds. The long-lived 

emission of these inorganic complexes is exploited for anisotropy experiments and the molecularlight 

switch nature of one of the probes will be extremely useful in assessing the conformational 

state of the protein. Finally we describe a family of peptides that can be used indirectly to label and 

monitor activation protein with these probes for further examination and to selectively activate the 

protein via one of its pathways. 

118


PB 

Conformational analysis of nucleoside analogue 

dideoxycytidine and comparison with the natural 

nucleoside deoxycytidine 

F. DÍEZ AND M. DE LA FUENTE 

Dept. of Ciencias y Técnicas Fisicoquímicas, Universidad Nacional de 

Educación a Distancia (UNED), c/ Senda del Rey s/n, E-28040, Spain, 

mfuente@ccia.uned.es 

A theoretical conformational analysis of the 2’,3’-dideoxycytidine (ddC) nucleoside analogue and a 

comparison with the natural nucleoside 2’-deoxicitydine (dC) was carried out. The potential 

energy scanning (PES) of both molecules by rotation of torsional angles χ (glycosidic bond) ,γ 

(C4´-C5´bond) and β (C5´-O5´ bond) was computed. These dihedral angles simultaneously were 

fixed at values varying between 0 and 360º in steps of 60º, all other geometrical parameters were 

relaxed during optimizations. The calculations were completed by using density functional 

methods including the Beck’s three parameters exchange functional (B3) in combination with the 

correlation functional of Lee, Yang and Parr (LYP). Further geometry optimizations, without any 

restriction, were accomplished for selected conformers, using the MP2 approach (Mφller-Plessett 

second-order perturbative method). The 6-31G(d,p) base set represents a compromise between 

accuracy and computational cost, therefore, this was the base set selected for these studies. All the 

calculations were performed by using the Gaussian 03 program package [1] running on 

Computational Chemistry Laboratory from Universidad Nacional de Educacion a Distancia 

(UNED) [2]. The energetic and geometrical data were analyzed, and energy distributions of 

different conformers as a function of the three scanned angles, as well as the sugar puckering 

(pseudorotational and maximum torsion angles) were evaluated. Syn/anti conformation 

distribution and North/South conformation populations were estimated. Although both 

nucleosides shown similarity in all the tested parameters, a very significant difference is found in 

deoxyribosa conformation. That is, while in dC the south conformation was predominant (86 %), 

most of the conformers show North puckering (87.5%) in ddC. Further studies will be realized to 

analyze these differences in condensed phase, closer to physiological conditions. 

References 

[1] Gaussian 03, revision B.0.4 and B.0.5 Gaussian INC ,Pittsburgh PA 2003. 

[2] Laboratorio de química computacional (Lab-QC), UNED, http://www.uned.es/lab-qc, 2004. 

119


PB 

Raman mapping of single gold nanoparticles in cells 

L. HARTSUIKER, R.G. RAYAVARAPU, W. PETERSEN, S. MANOHAR, T.G. VAN LEEUWEN AND C. 

OTTO 

Biophysical Engineering Group, University of Twente, P.O. Box 217, 

7500 AE, Enschede, The Netherlands 

Gold nanorods are rapidly emerging for use in biomedical applications due to their 

biocompatibility and their favourable optical properties, such as an enhanced absorption crosssection. 

The latter is a result of surface plasmon resonance bands (SPB). These bands can be tuned 

to the near-infrared region (NIR) by changing the aspects ratio of the rods. Tissue is relatively 

transparent in the NIR, enabling increased penetration depths for NIR optical imaging. 

Conventional tumour imaging suffers from low contrast with respect to the surrounding tissue, 

but by administering gold nanoparticles with NIR-SPB to the tumour site, high contrast noninvasive 

cancer imaging can be achieved, which is essential for diagnostics and treatment of early 

stage carcinomas. CTAB (cetyl trimethylammonium ) so far seems to be an essential surfactant for 

the production of gold nanorods. CTAB is however highly cytotoxic and particles are therefore 

subjected to surface modification before administration to cells. Rods treated with methoxy- 

(polyethylene glycol)-thiol (mPEG-SH) appear completely nontoxic. The aggregation of metal 

nanoparticles is a well known phenomenon. Aggregated metal nanoparticles have complex optical 

properties, which seem to be related to the precise composition, extent and size of the aggregate. 

The Raman signals of non-aggregated gold nanorods in solution are measured before and after 

PEGylation. The Raman spectra contain various signals characteristic for broad-background 

fluorescence-like emission and narrow-band characteristic Raman features of surface-adsorbed 

molecules. Raman images are made to monitor the interaction of gold nanorods with cells. Gold 

nanorod detection in cells may be facilitated by the use of dyes as Raman markers. We have 

explored the use of indocyanine green (ICG), which is an FDA-approved dye for clinical 

applications. Extremely intense Raman spectra can be acquired, which will eventually result in 

molecular diagnostic intracellular particles. Other biomedical applications can be foreseen. We will 

discuss the relation of the molecule-to-particle ratio to the intensity of the Raman scattering. 

120


PB 

Tautomerism of the most stable conformers of analogue 

D4T and natural Thymidine nucleosides. Influence of 

an aqueous environment using MP2 and DFT methods. 

M. ALCOLEA PALAFOX AND N. IZA 

Dept. de Química Física I, Universidad Complutense, Ciudad 

Universitaria, Madrid, 28040, Spain 

The energies of the most stable conformers of the anti-HIV agent D4T (stavudine) and Thymidine 

(Thy) nucleosides were calculated in a previous work [1]. In the present communication, a 

comparative theoretical conformeral analysis of the tautomerism of the two most stable 

conformers of D4T and Thy was carried out, by using the B3LYP and MP2 quantum chemical 

methods with the Gaussian 03 package [2]. For each conformer only two stable enol forms T3 and 

T5 were obtained when considering different positiones of hydrogen around the base. The 

calculated structure data and energy values for both nucleosides in these tautomers were 

compared to the T1 keto form as reference (see Table). Relative stabilities of all tautomers were 

established. Tautomer T3 appears always more estable than T5. Also T3 appears more stable in the 

natural nucleoside Thy than in D4T. The effect of water on the tautomers was estimated by an 

explicit number of water molecules surrounding the nucleoside up to five, and on the other hand 

by the Tomasi’s Polarized Continuum Model (PCM). A total of about 200 clusters were optimised 

and the geometrical parameters and energies discussed. The deformation and interaction CPcorrected 

energies [3] between the nucleoside and water molecules were determined. The 

microhydrated environment stabilizes remarkably the enol forms more than the canonical keto one, 

although the latter continues being as the most stable. Further increase in the number of water 

molecules reinforces the tautomerism. The changes in the intramolecular H-bonds and in the total 

atomic charges with hydration were discussed, showing an increase of the reactivity on the O2 and 

O4 carbonyl atoms. 

Table . Relative energies (in kcal/mol) calculated in the three tautomers of the two most 

stable conformers of the D4T and Thy molecules. 

D4T Thy 

Conformer I Conformer II Conformer I Conformer II 

Level of theory T1 T3 T5 T1 T3 T5 T1 T3 T5 T1 T3 T5 

Isolated state 

B3LYP/6-31G** (+ZPE) 

B3LYP/6-311++G(3df,pd) 

MP2/6-31G** 

Water solution (PCM) 

B3LYP/6-31G**(+ZPE) 

B3LYP/6-311++G(2d,p) 

0 

0 

0 

0& 0& 12.68 

12.76 

13.25 

10.05& 9.79& 20.34 

19.59 

20.16 

15.50& 14.18& 0 

0 

0 

0 

0 & 

121 

12.50 

12.51 

13.00 

9.84 

9.85 & 

20.63 

20.00 

20.43 

15.56 

14.44 & 

0 a 

0 b 

0 c 

0 d 

11.52 

12.31 

11.64 

(n) 

20.14 

19.63 

19.98 

(n) 

0 e 

0 f 

0 g 

0 h 

12.36 

12.46 

12.95 

&Saddle point. a -874.903915 AU. b –875.458056 AU. c –872.671789 AU. d -874.903912 AU 

e -874.905374 AU. f –875.458447 AU. g –872.673866 AU. h -874.946535 AU. (n) not stable 

References 

[1] M. Alcolea Palafox, N. Iza, M. de la Fuente, and R. Navarro, J. Phys. Chem. B, 113, 2458-2476 (2009). 

[2] Gaussian 03, Revision B.04, Gaussian Inc., Pittsburg, PA (2003). 

[3] V. I. Danilov, T. van Mourik, and V. I. Poltev, Chem Phys. Letters, 429, 255-260 (2006). 

9.87 

20.29 

19.67 

20.10 

15.32


PB 

Single turnover event detection of chymotrypsin catalysis 

T. TERENTYEVA 1 , G. DE CREMER 1 , K. BLANK 2 , M. B.J. ROEFFAERS 1 AND J. HOFKENS 1 

1. Dept. of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium 

2. Inst. for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ 

Nijmegen, The Netherlands 

Chymotrypsin is an abounded enzyme in nature. The chemical mechanism of its catalysis is well 

understood, however, enzymes are dynamic molecules which exert structural fluctuations. The 

influence of enzyme’s dynamics on the kinetics of their catalysis still remains the challenging 

question in enzymology. Fluorescence based single-molecule experiments enable to overcome 

ensemble averaging, to get detailed mechanistic insight in catalysis and to follow conformation 

changes. There is a wide range of single-molecule approaches to study catalytic activity of 

enzymes. Their main differences are the time resolution and the time window of the experiment. 

One of the sophisticated tools that allow the detection of individual fluorescent reporter molecules 

changing their properties in the enzymatic reaction during the time is confocal fluorescence 

microscopy. The direct and real-time observation for extended periods of time of individual 

chymotrypsin enzymes immobilized in an agarose gel is performed with this technique. To follow 

single enzyme turnovers we use the conversion upon enzymatic hydrolysis of a non-fluorescent 

substrate, rhodamine 110 bis-(suc-Ala-Ala-Pro-Phe), into a strongly fluorescent product, 

rhodamine 110. Analysis of the recorded single turnover time traces is done in two different ways. 

In the first method successive enzyme turnovers are counted by thresholding the binned 

fluorescence intensity time trace to distinguish signal from noise and applying the on- and off-state 

events. Plotting off-times into histogram shows that its probability density function exhibits a 

stretched exponential behavior. This refers to the fluctuations in the rate constants and 

corresponds to the conformational changes. In the second method the autocorrelation function of 

the intensity is calculated directly on photon arrival times. The data in the relevant time scale (0,1 – 

10 s) does not fit mono exponential decay neither. We have investigated the response of the singleenzyme 

activity on pH-increase. Upon addition of base to the substrate solution, we have observed 

a gradual decrease of enzyme activity without emphatic changes in dynamics. Though, not all 

individual enzymes respond similarly to the applied alteration and moreover, there is a slight 

spread of the pH-optimum. Thus, for the first time we prove that the concepts of dynamic and 

static disorder can be applied not only to activity, but also to deactivation processes of individual 

enzymes. 

References 

Fig. 1 – Confocal setup and data processing of single turnover transient trace. 

[1] Velonia,K.etal.Angew.Chemie-Int.Ed.44,560-564(2005). 

[2] DeCremer,G.etal.JACS129,15458-15459(2007). 

122


PB 

Determination of physical properties of a single peptide 

by AFM experiments 

M. VASSALLI 1,2 , F.SBRANA 2 , B.TIRIBILLI 2,3 , B.BOCHICCHIO 4 , A.M.TAMBURRO 4 

1. IBF – CNR ; Genoa (GE) ; Italy 

2] CSDC – Physics Dept. ; University of Florence ; Italy 

3] ISC – CNR ; Sesto Fiorentino (FI) ; Italy 

4] Chemistry Dept. ; University of Basilicata ; Potenza ; Italy 

By stretching a polymer in solution [1] using single molecule techniques it is possible to infer about 

its physical properties. In particular, AFM stretching experiments allow for a full characterization 

of the elasto-mechanical properties of the sample under study, taking into account both statical 

and dynamical regimes [2]. In the presented work, single molecule AFM force spectroscopy 

experiments have been used to determine mechanical properties of a polymer obtained starting 

from the Exon 28 (Ex28) of the human elastin gene. Elastin is a protein with important mechanical 

properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of 

many hydrophobic unstructured domains (such as Ex28) into the protein structure. The Ex28 

coded polymer has been used as a starting point to obtain bio-materials with specialized elastomechanical 

functions. In particular, a mutated polypeptide based on the EX28 sequence has been 

synthesisized (named EX28K) with the aim of obtaining a new polymer with the same mechanical 

and physical properties of the native molecule but with increased aggregation properties, induced 

by a cross-linking reaction. AFM stretching experiments have been used to verify the mechanical 

properties of the engineered proteins at a single molecule level. The obtained results allowed us 

not only to answer this question, but also to give some insight into the first aggregation steps of the 

polymer towards the formation of reticulated structures. 

References 

Fig. 1 – single molecule AFM stretching experiments on EX28K 

coded polymer after cross-linking 

[2] C.Bustamante, J.F.Marko, E.D.Siggia, S.Smith; Science 265, 1599 – 1600 (1994) 

[2] R.A.Harris, J.E.Hearst; J. Chem. Phys. 44, 2595 (1966) 

123


PB 

Optical tweezers assisted dynamic force spectroscopy 

on single receptor/ligand contacts 

C.WAGNER, M. SALOMO, F.KREMER 

1. Dept. of Experimental Physics 1, University of Leipzig, Linnéstraße 5, 

Leipzig, D-04103, Leipzig 

The extraordinary features of optical tweezers having a nm- resolution in positioning a micronsized 

colloid and an accuracy of (±50 fN) in measuring the forces acting on it enable one to study 

the interaction within a single receptor/ligand-contact. To establish a model system, the 

interaction between Protein A from Staphylococcus aureus and Immunoglobulin G from rabbit 

serum (RIgG) is investigated (Fig. 1). It is demonstrated that the rupture forces depend on the 

loading rate. This effect is well known in the literature and the data obtained were found to be in 

good agreement with an already published theoretical model [1]. By use of this model, the off-rate 

at zero force is determined. The comparison of the off-rates from Immunoglobulin G molecules 

from rabbit, human and bovine serum, each binding to Protein A, yields different values. These 

can be related to the varying affinities measured with bulk methods. Our experimental setup can 

probe the interaction between a single receptor and its specific ligand under changing conditions 

and hence offers manifold applications in single molecule biotechnology. 

References 

Fig. 1 – Force-Distance trace. Inset: Scheme of the experimental 

setup. For investigating the interaction between Receptor and 

Ligand, the particular molecules are immobilized on the surface of 

microparticles. 

[1] M. Salomo, U.F. Keyser, and F. Kremer, <strong>European</strong> Biophysics Journal 37, 927-934 (2008) 

124


PB 

Development of Raman microspectrometer for timecourse 

imaging of single living cells 

A. ZOLADEK, F. PASCUT AND I. NOTINGHER 

Nanoscience Group, School of Physics and Astronomy, University of 

Nottingham, University Park, NOTTINGHAM, NG7 2RD 

Cells are the smallest units of life and structural components of all living organisms. Nearly all 

diseases arise from cellular abnormalities that originate in biochemical intracellular changes. 

Therefore studying cells behavior, metabolism and biochemistry is an important area of current 

research. A compelling technique for studying live cells is Raman microspectroscopy. Raman 

spectroscopy is extremely information rich. It has many advantages when used to study biological 

samples as there is no requirement of special sample preparation, it is non-invasive and has high 

chemical specification. However the unique advantage of Raman microspectroscopy is ability to 

provide the biochemical information in non-invasive way therefore it allows time-course 

experiments on single living cell, which no other techniques can offer. The major aim of our work 

was to construct an inverted Raman micro-spectrometer with the ability to perform multi-hours 

spectral measurements and minimal possible intervention in biochemical processes of cells. The 

choice of laser wavelength and power is crucial when dealing with biological materials, as it has to 

allow the collection of good quality Raman spectra in short time and avoid inducing cell damage 

or autofluorescence, which could hinder the much weaker Raman Effect. For these reasons the 

laser chosen falls in the near-IR region. Our system allows point measurement as well as high 

quality Raman maps of cells with micrometer spatial resolution. The setup has the ability to 

perform fluorescence imaging of cells in several filter setups. Therefore it is possible to accomplish 

a Raman map of the live cell and then after obtain fluorescence image of the molecules of interest 

on the same sample. Incorporating this well-known technique in cell biology into the Raman 

system will allow correlation between Raman spectroscopic changes and biological activity of cells. 

To maintain feasible conditions and ensuring cell viability for several hours of measurement, an 

inverted microscope was chosen enclosed in an incubator to provide the appropriate temperature 

and CO2 stability. The use of the inverted Raman micro-spectrometer offers more flexibility for 

performing Raman spectral imaging and time-course experiments on live cells, since cells are not 

disturbed during experiments and issues related to bacterial contamination are avoided. Several 

measurements of cells were applied by both point measurement and by Raman spectral imaging 

maps of cells on our system to show the potential of Raman techniques in cell biology studies; this 

included experiments of apoptotic cells after application of anticancer drug or measurements of 

junctions between two cell types. 

125

Abramczyk H. 61, PA-37, PB-33 

Ackermann K.R. 86 

Adnan N. PB-41 

Afseth N.K. 73, PB-107 

Ahmad S. PA-20 

Akyuz S. PB-70, PB-79 

Alcolea Palafox M. 75, PB-52, PB-121 

Alenkina I.V. 60 

Alessi M. PA-21 

Alexeeva I. PA-111 

Alexiev U. 27 

Alieva R. 76 

Almog O. PB-29 

Amado A.M. PA-29, PA-103 

Ami D. PA-22, PB-105 

Ammenti A. 100 

Andersen K.B. 62 

Andreeva A. PA-23 

Antonicelli F. 90 

Anzenbacher P. PB-48 

Apostolova I. PA-23 

Arai T. PB-34 

Araiza-Reyna E.A. PA-24 

Araujo-Andrade C. PA-24, PB-2, PB-17 

Arazam N. PB-1 

Arcovito A 12 

Ardiccioni C. 12 

Arenas J.F. PB-60 

Argov S. PA-86 

Arndt M. PB-61 

Arrondo J.L.R. 59 

Arunmurthhy T.V.S. PB-52 

Atrian S. PB-88 

Astilean S. PA-25 

Atta D. 78 

Audebrand M. PB-113 

Auxina L. PB-18 

Avila-Donoso H. PA-24 

Avila Rodriguez R. PB-2 

Ayala A.P. PA-82 

Babu N.S. PA-55 

Badache A. PA-5 

Bagheri-Garmarudi A. PA-26 

Baia M. PA-25 

Balsamo A. PB-36 

Banerjee S. PB-63 

Bar I. PA-27 

Baranska M. PA-28 

Barko Sz. 44 

Barth A. 43 

Barthélémy P. PB-1 

Bartucci R. PB-85 

Batabyal D.A. PB-92 

Batista de Carvalho LAE PA-29, PB-21 

Baumruk V. 46, PA-98, PB-102 

Becker S. PB-90 

Beckford G. 88 

Bednarova L. 46 

Bedotti R. PB-39 

Bedzinski R. PB-20 

Bekhradnia A.R. PB-64 

Belagyi J. 44 

AUTHOR INDEX 

xv 

Beleites C. PB-3, PB-6 

Bell D. 89 

Bellavia G. PB-65 

Belogurova N. 76, PA-72 

Belov A.S. PB-55 

Belshi A. PA-80 

Benard A. 81, PB-4, PB-9 

Benedetto A. PA-17 

Benner D. 83 

Bergoin M. PA-110 

Bernard P. 63 

Bernhard G. PA-76 

Berthault P. PB-7 

Bethoin K. PB-16 

Bhattacharya K. 79 

Bian N. 89 

Bichenkova E.V. 32 

Bilbao P. 59 

Binev I.G. PA-95 

Bisby R.H. PB-5 

Bittl R. PA-122 

Bizzarri A.R. PA-30 

Blackledge C.W. PA-31 

Blanchard J. PB-100 

Blanchet L. 71 

Blank K. PB-122 

Blaszczack Z. PA-32 

Blazka S. 89 

Bligny R. PB-31 

Boca S. PA-25 

Bochicchio B. PB-123 

Bocklitz T. 86, PA-119, PB-28, 

PB-116 

Bode B.E. PA-3 

Boffi A. 13, PA-47, PA-78 

Bogomolny E. PA-86 

Böhme R. PB-117 

Böhme S. PA-1 

Bonaccorsi L. PB-58 

Bonarek P. PA-53, PB-66 

Bonifacio A. PB-6 

Bonnier F. 79, PA-15, PB-27 

Bonura M. PB-67 

Botchway S.W. PB-5 

Boulard Y. PB-7 

Bour P. PA-98 

Boutin C. PB-7 

Boyarkin O.V. 23, PA-87 

Brault D. PB-69 

Breer K. PA-33 

Brenowitz M. PB-100 

Bridelli M.G. PB-39 

Brondino C. PA-34 

Brotin T. PB-7 

Brown L. PB-71 

Brozek-Pluska B. 61, PB-33 

Brucker S. PB-68 

Brumas V. PB-14 

Bruni F. 39 

Brunori M. 12 

Buchwald T. PA-32 

Buck D.P. PB-41

Buck S. PA-2 

Bulone D. PB-103 

Buriankova L. PB-69 

Bursakov S.A. PA-34, PA-67 

Bürstel I. PA-61 

Buzova D. PB-69 

Byrne H.J. 79, PA-12, PA-15, 

PB-27, PB-30 

Bzowska A. PA-33, PB-115 

Cakic M. PA-83 

Caldeira C.J. PB-42 

Callens F. PA-9 

Calvete J.J. PA-67 

Cammarata M. 37 

Campbell C.J. PB-59 

Cannas M. PA-41 

Cannistraro S. 14, PA-30 

Canpean V. PA-25 

Capdevila M. PB-88 

Capelletti R. PB-39 

Capozzi F. PA-7 

Capozzi V. PA-35 

Cappugi G. PB-112 

Cardot-Leccia N. 63 

Carelli C. 72, PA-58 

Carmona P. PB-50 

Carravetta V. 41 

Carriere M. PB-7 

Carrotta R. PB-96 

Carsughi F. PA-124 

Caryn S. PB-16 

Casella M. PB-8 

Castro A. PA-35 

Castro C.M. PB-56 

Castro J.L. PA-77, PB-60 

Casu 101 

Ceccarelli 101 

Cecconi F. 100 

Celik S. PB-70, PB-79 

Cerf E. 58, PB-93 

Chamoun J. PB-71 

Champion P.M. 19 

Chatgilialoglu C. PB-88 

Chavarha M.M. PA-113 

Chen S.H. 8 

Cheng H. PB-100 

Chi H. PB-99 

Chikhirzhina E. 95 

Chikvaidze E. 55, PB-104 

Chirico G. 80 

Chorvat D. Jr. PB-69 

Chowdhury P. PA-36 

Chturvedi D. PA-82 

Ciacka P. 61, PA-37 

Cinta-Pinzaru S. 85, PB-13 

Citron S. PA-74, PB-25 

Clark I.P. 92 

Clark P.L. PB-115 

Clement J.H. PB-28 

Clivio P. 94 

Coelho C. PA-38 

Cohen A. PB-38 

xvi 

Cohen-Luria R. 87, PB-29 

Coic Y.M. 72, PA-58 

Colette Y. PA-5 

Colindres M. PA-39 

Collini M. 80 

Collins J. G. PB-41 

Conti F. 31 

Conti J. PB-16 

Conti Nibali V. PA-6, PB-72 

Cooke J. PB-71 

Cordone L. 9, PA-51, PA-92, PB-65 

Coreno M. 41, PA-125 

Corsaro C. PA-6 

Cosgrave L. PB-118 

Cotoni K. 89 

Cottone G. PA-51, PA-92, PB-65 

Crisostomo A.G. PB-5 

Crupi C. PB-72 

Cupane A. PA-112, PB-65, PB-67 

Czarnik-Matusewicz B. PA-40, PB-53 

D’Alfonso L. 80 

D’Amico M. PA-41, PB-97 

D’Angelo G. PA-6, PB-72 

Da Silva J.B.P. PB-21 

D’Auria S. 57, PB-62, PB-110 

David C. PB-73 

Das T. 42 

Dastvan R. PA-3 

De Angelis F. PA-89 

De Antoni G. PA-24, PB-17 

De Chaumont C. 49 

Deckert V. PA-73, PB-117 

De Coninck J. PB-16 

De Cooman H. PA-9 

De Cremer G. PB-122 

De Fries N. 62 

De Juan A. 71 

Dehelean C. 85, PB-12, PB-13 

De llanos R. PA-42, PA-106 

De la Arada I. 59 

De La Fuente M. PB-119 

Dellago C. 16 

De Marco A. PB-105 

Dembska A. 93 

Dennison C. PB-98 

Derclaye S. 58 

Derenne A. 81, PA-49, PB-9 

Desbat B. PB-1 

De Simone M. 41 

Desmedt C. PB-4 

Desvaux H. PB-7 

Dettin M. PB-10 

Devocelle M. PB-118 

Devouge S. PB-16 

Dgebuadze M. PB-104 

Dhamelincourt M.C. PA-43 

Dicko C. 54 

Diez F. PB-119 

Di Fabrizio E. PB-8 

Di Carlo M. PB-96 

Di Foggia M. PB-10 

Diller R. 22, PA-39, PA-45,

PA-121 

D’Inca H. PB-11, PB-15 

D’Ischia M. 31, PA-60 

Dlouha H. 46 

Dobrowolski J. Cz. PA-28, PA-44 

Dockter C. 6 

Doglia S.M. PA-22, PB-105 

Domenech J. PB-35 

Dominaci F. PA-30 

Domingo C. PA-42, PA-106 

Domreatcheva T. PA-121 

Doorley G.W. 92 

Dovbeshko G. PB-43 

Draux F. 91 

Drescher D. 82 

Dribinsky B. PB-43 

Droghetti E. 13 

Ducic T. PA-123 

Dudas R. PB-87 

Dufrene Y. 58 

Dumas P. 91 

Durbecq V. PB-4 

Durchschlag H. PB-94 

Durlach A. 63 

Dutasta J.P. PB-7 

Dziedzicka-Wasylewska PA-53, PB-66 

Dziuk M. PB-94 

Eaton P. PB-42 

Ebrahimzadeh M.A. PB-64 

Egawa T. 42, PB-92 

Egger D. PB-90 

Ehmer D. PA-45 

Eichler S. PB-74 

Enderlein J. 78 

Enescu M. PB-73 

Engdhal A. 62 

Engelhard M. 22 

Engels J.W. PB-44 

Engelsen S.B. PA-7 

Erdmann S. PA-119 

Eremin V.V. PB-55 

Eremina N. 43 

Esimbekova E. PA-88 

Espinoza Herrera SJ. PA-46 

Ettrich R. PB-77 

Fagnano C. PB-10 

Fahmy K. 27, PB-74 

Fajer P. PB-71 

Falamas A. 85, PB-12, PB-13 

Fanelli D. PB-112 

Farcau C. PA-25 

Fares C. PB-90 

Farhi E. PB-31 

Fas B.A. PB-54 

Feis A. 13, PA-47, PA-78 

Feracci, M. PA-5 

Fernandes A.P. PA-116 

Ferreri C. PB-88 

Fersht A. 3 

Fesenko O. PB-43 

Feyer V. 41, PA-125 

Fiallo M.M.L. PB-14 

xvii 

Fitter J. 78 

Flaender M. PB-45 

Flamigni A. PB-6 

Floch P. PB-15 

Foerstendorf H. PA-76 

Foerster U. PB-44 

Foggi P. PA-78 

Fomina M.G. PA-48 

Fonin A.V. PB-62, PB-95 

Formaggio F. 34 

Forster R.J. 49, PA-31, PB-118 

Forte C. PA-4 

Foster S. PA-85 

Fouquet P. PB-76 

Franzen S. PA-90 

Frausto-Reyes C. PA-24, PB-17 

Freddi G. PB-34 

Freddi S. 80 

Friedman J.M. 5 

Friedman N. 22 

Friedrich B. PA-61, PA-122 

Frielingsdorf S. PA-122 

Frosch T. 64 

Fuchs P. PA-57 

Fucina T.Y. PA-117 

Fuller F. PA-13, PA-14 

Funk M. PB-90 

Fürtig B. PA-2 

Gaiddon C. PB-47 

Gambarelli S. PB-45 

Gandolfi M.G. PB-24 

Garagna S. PA-22 

Garasevych S. PA-62, PA-111 

Garcia-Ramos J.V. PA-42, PA-106 

Gasior-Glogowska M. PB-20 

Gasparutto D. PB-45 

Gasper R. 81, PA-49, PB-9 

Gavare M. PB-18 

Gavel O.Yu. PA-34, PA-67 

Gdalevsky Ga. Y. PB-29 

Gebrezgiabher M.B. 32 

Gellini C. PA-47, PA-78 

Gellner M. 84 

Gennis R.B. PB-83 

Gepshtein R. 48 

Gerasimova M.A. PA-48, PA-50, PA-69 

Gershanik A. 87 

Gerwert K. 38, PA-70, PB-68 

Ghasemi K. PA-26 

Gheber L.A. PA-27 

Ghomi M. 72, PA-58 

Giacomazza D. PB-96 

Gilch P. 94 

Giordano F.M. PB-97 

Girstun A. PA-33 

Giuffrè A. 12 

Giuffrida, S. PA-51, PA-92, PB-65 

Glaubitz C. 30 

Gloria D. PA-85 

Glotova O. PA-11 

Glusic M. PA-52 

Gnatiuk O. PB-43

Gobinet C. PA-105 

Goldgur Y. PB-29 

Goldsztein A. PB-16 

Golebiowski F. PA-53 

Gomez-Zavaglia A. PA-24, PB-17 

Gonzalez M.A. PA-17 

Gonzalez Amaro A. PB-2 

Gonzalez Velasco J. 59 

Goormaghtigh E. 58, 81, PA-49, PB-4, PB- 

9, PB-16, PB-93, PB-101 

Gorecki A. PA-53, PB-66 

Goris T. PA-122 

Gouveia Z. PA-67, PA-116 

Gramatica F. PB-8 

Grdadolnik J. PA-52 

Gregor J. 78 

Greve T.M. 62 

Griesinger C. PB-90 

Gromova M. PB-31 

Gross R. 22, PA-39, PA-45, 

PA-121 

Grosserüschkamp M. PB-75 

Grosso M. PB-36 

Grube M. PB-18, PB-19 

Gruebele M. PB-90 

Grzelakowski M. 78 

Gruccione A. PA-47 

Gruenewald C. PB-44 

Grunbeck A. PB-63 

Guerlesquin F. PA-5 

Guerrero Martinez A. 75 

Guidi M. 23 

Guidoni L. 99 

Gulic R. PB-90 

Gulnov D. PA-88 

Gutberlet T. PA-19 

Guyot G. PA-38 

Guzzi R. PB-85, PB-98 

Hagarman A. 53, PA-4, PA-21, PA-54 

Hajdukova-Smidova N. PA-97 

Haliloglu T. PB-54 

Hall S.A. 24 

Hamley I.W. PA-101 

Hamm P. 4 

Handgraaf J.-W. 34 

Hansberry D.R. PA-55 

Hanuza J. PB-20 

Harauz G. PB-84 

Haris P.I. PA-56 

Harlepp S. PB-47 

Hartsuiker L. PB-120 

Haslinger P. PB-61 

Hastings G. PA-57 

Haupt K. PA-27 

Hauser K. 40 

Havlicek V. 65 

Hayes S.C. 70 

Hebraud P. PB-47 

Hedge S. PB-100 

Heidary N. PA-122 

Heisler I.A. 20 

Helios K. PB-23 

xviii 

Henary M. 88 

Henkel T. 86 

Hennemann L.E. 83 

Hennig M. PB-76 

Hering J.A. PA-56 

Hermann G. PA-39 

Hermanowicz K. PB-20 

Hermelink A. PA-73, PB-26 

Hernandez B. 72, PA-58 

Hibbert D.B. PA-85 

Hild G. 44, PB-87 

Hildebrandt P. PA-61, PA-107, PA-122, 

PB-82 

Hilger D. 6 

Hilliard J. PA-57 

Hincha D.K. PB-109 

Hofbauerova K. PB-77, PB-102 

Hoffmann S.V. 54 

Hofkens J. PB-122 

Homblé F. PB-16 

Horch M. PA-122 

Hore D.K. 24 

Howe O. PB-30 

Howell M. PB-71 

Howes B. D. 13, PA-90 

Hsu Y. PA-57 

Huang R. 40 

Huber G. PB-7 

Hubert Joe I. PA-59, PA-99 

Huijser A. PA-60 

Huleihel M. 77 

Hummel P. PA-61, PA-122 

Hundertmark H. PB-109 

Huppert D. 48, PB-89 

Hushcha T. PB-78 

Iakhnenko M. PA-62, PA-111 

Ihee H. 7 

Iosin M. PA-25 

Itkin A. 58 

Ivanov-Tzonchev R. PB-17 

Iza N. PB-121 

Jablonska J. 61, PB-33 

Jacobs R.M.J. PB-76 

Jadzyn M. PA-8, PA-11 

Jain R. PB-57 

Jaiswal S. PA-63, PA-109 

Jamin N. PB-7 

Jamroz M.H. PA-44 

Jancura D. PB-69 

Jariashvili K. 55, PB-104 

Jeannesson P. PA-105 

Jelovica Badovinac I. PA-78 

Jena K.C. 24 

Jeschke G. 6 

Ji H. 42 

Joseph V. 82 

Juhna T. PB-19 

Jung H. 6 

Jung Y.M. PA-40 

Juranyi F. PA-19 

Juskowiak B. 93 

Kalinowska M. PA-75, PA-100

Kalisky Y. 87 

Kambara O. PA-64 

Kandori H. 29 

Kantarovich K. PA-27 

Karjalainen E.-L. 43 

Karolin J. PB-5 

Käs J. PB-116 

Kasyanenko N. PB-43 

Katranidis A. 78 

Kawaguchi S. 29 

Keane P. 92 

Kecel S. PB-70, PB-79 

Keiderling T.A. 40, 56, PB-99 

Kejnovska I. PB-46 

Kelly J.M. 92 

Keyes T.E. 49, PA-31, PB-118 

Khanmohammadi M. PA-26, PA-65 

Khatypov R.A. PA-117 

Kheiro-Derfoufi M. PA-89 

Khiati S. PB-1 

Khrapunov S. PB-100 

Kierdaszuk B. PA-66 

Kim S.H. 88 

Kiss R. PA-49 

Kladova A.V. PA-34, PA-67 

Klajner M. PB-47 

Klare J.P. PA-1 

Klein I. PB-32 

Klein O. PB-32 

Kleiren E. PB-101 

Kneipp J. 82, PA-104 

Knief P. 79, PA-12 

Knoll W. 45, PA-18, PB-75, PB-83 

Kobayashi T. 69 

Kobielarz M. PB-20 

Kocisova E. PA-68, PA-96 

Kogan A. PB-29 

Kohler A. 73, 91, PB-107 

Kohoutova J. PB-77 

Kolos R. PA-44 

Kominis I. PB-80 

Komorowska M. PB-20, PB-53 

Kondo M. 20 

Koning V. 26 

Kopecky V. Jr. PB-77, PB-102 

Kordek R. 61, PB-33 

Koshkov K.V. PA-69 

Kostyleva E. 95, PB-95 

Kothe E. PA-119 

Kotlyar S. PB-89 

Kötting C. PA-70, PB-68 

Kourkoumelis N. PA-71 

Kowalczyk E. PB-66 

Kozielski M. PA-32 

Krafft C. PB-13, PB-28 

Krasowska J. PB-115 

Kratasyuk V.A. PA-88 

Krejtschi C. 40 

Kremer F. PB-124 

Kroh L.W. PA-104 

Kruppa G. 65 

Kruszewski M. PB-49 

xix 

Kubanova M. 46 

Kubon J. 94 

Kudryasheva N. 76, PA-72 

Kumar H. 43, 75, PB-52 

Kumar M. PA-63 

Kumar S. 43, PA-109 

Kupi T. 44 

Kursula P. 45, PA-18 

Kustner B. 84 

Kuznetsova I.M. 57, PB-62, PB-95, 

PB-110, PB-114 

Kypr J. PB-46 

L’Abbate N. PA-35 

Laczko-Dobos H. PB-86 

Lakhani A. PB-99 

Lakomek N.A. PB-90 

Lamparter T. PA-45 

Langer K. PB-37 

Larsen N.W. 62 

Larsimont D. PB-4 

Lasalvia M. PA-35 

Lasch P. PA-73, PB-26 

Laskova B. PA-84 

Lau K. PA-73 

Laurent A. PA-74, PB-11, PB-15, 

PB-25 

Lee D. PB-90 

Lehene C. 85 

Leibl W. 71 

Lemr K. 65 

Lendzian F. PA-122, PB-82 

Lenferink A. 74 

Lenz O. PA-61, PA-122 

Leone M. PB-106 

Leteurtre F. PB-7 

Levantino M. PA-112 

Lewandowski W. PA-75, PA-100 

Lewis A. PA-74, PB-25 

Lewis K.L.M. PA-13, PA-14 

Lewis-Ballester A. PB-92 

Li B. PA-76 

Li L. 22 

Lima J.L.F.C. PB-56 

Lin J. PA-45 

Lin Y. PB-92 

Liu J. PB-105 

Loidl A. PB-90 

Lombardo D. PB-58 

Londer Y.Y. PA-116 

Longo A. PA-92, PB-58, PB-97 

Lopez Gonzalez J.J. PA-79 

Lopez-Ramirez M.R. PB-60 

Londero A. PA-24, PB-17 

Lu C. PB-92 

Lubitz W. PA-122, PB-82 

Lucio M. PB-56 

Lucotti A. PB-8 

Ludwig M. PA-122 

Lukacs A. PB-81 

Lunkenheimer P. PB-90 

Ly E. 63, PA-105, PB-11 

Lyng F.M. 79, PA-12, PA-15,

PB-27, PB-30 

Maccarini M. PB-76 

Machado N.F.L. PA-77 

Maczka M. PB-20 

Madathil S. 27, PB-74 

Mäder J. PA-104 

Maerz A. 86 

Magazù S. PA-17 

Maher W. PA-85 

Maksymowicz K. PB-20 

Mainreck N. 90 

Malon P. 46 

Malsch D. 86 

Manfait M. 63, 90, PA-74, PA-105, 

PB-11, PB-15, PB-25 

Mangione M.R. PB-103 

Maniu D. PA-25 

Manno M. PA-41, PB-97, PB-111 

Manohar S. PB-120 

Mäntele W. 30, PB-22, PB-32, 

PB-37, PB-91 

Marcelli A. PA-78 

Marchal A. PA-79 

Marchand-Brynaert J. PB-16 

Margitich M.O. PA-113 

Marksteiner M. PB-61 

Marini U. 100 

Mariani P. PA-124 

Marquez F. PA-79 

Marques M.P.M. PA-77, PB-21 

Martens H. 73 

Martinez J.R. PB-2 

Martinez-Mendoza J.R. PB-17 

Martorana V. PA-41, PB-97, PB-103, 

PB-111 

Marsich E. PB-6 

Marzocchi M.P. PA-80 

Masek V. PB-48 

Masino L. 11 

Matschulat A. 82 

Matthieu D. PA-54 

Maurer J. PB-22 

Maurya M.K. PB-40 

Mazzarella L. PB-36 

McElheny D. 56 

McNally A. 49 

McQuillan A.J. PA-93 

Meade A.D. 79, PA-12, PA-15, PB-27 

Measey T.J. 53, PA-4, PA-54 

Meczynska-Wielgosz S. PB-49 

Meech S.R. 20, PB-81 

Meier B.H. PA-4 

Meier W. 78 

Meiler J. PB-90 

Meixner A.J. 83 

Mendes C.S.M. PB-21 

Mereghetti P. PA-22 

Merlino A. PB-36 

Mermet A. PA-110 

Methenitis C. PB-14 

Metreveli N. 55, PB-104 

Meyer S. PA-1 

xx 

Mezzenga E. PA-35 

Mezzetti A. 71, PA-43 

Michalska D. PB-23 

Migliardo F. PA-17 

Mijatovic T. PA-49 

Milder O.B. 60 

Milecki J. PA-8 

Miles S.M. 32 

Militello V. PB-106 

Millo D. PA-122, PB-82 

Millot J.M. PA-74, PB-25 

Milov A.D. 34 

Mironova N.L. 32 

Mironova-Ulmane N. PA-81 

Mishra S. PA-82 

Miskovsky P. PB-69 

Mitic Z. PA-83 

Mobili P. PA-24, PB-17 

Modena E. PB-24 

Mojzes P. PA-84, PA-96, PB-48, 

PB-51 

Molina M. PB-50 

Mondelli C. PA-17 

Montejo M. PA-79 

Monti S. PA-4 

Moran G. PA-85 

Moran N. 49, PA-31, PB-118 

Mordechai S. 77, PA-86 

Moreh R. PA-86 

Moschetti T. 12 

Moura I. PA-34, PA-67 

Moura J.J.G. PA-34, PA-67 

Mroginski M.A. PB-82 

Mukamel S. PA-16 

Myers J.A. PA-13, PA-14 

Nagornova N.S. 23, PA-87 

Nagy A. PB-87 

Nakasone Y. 21 

Nallet F. PB-1 

Namicheishvili L. 55, PB-104 

Namur J. PA-74, PB-11, PB-15, 

PB-25 

Napolitano A. 31, PA-60 

Narayanaswami V. PB-93 

Natalello A. PA-22, PB-105 

Natali F. 45, PA-18 

Naumann D. PA-73, PB-26 

Naumann R.L.C. PB-75, PB-83 

Navailles L. PB-1 

Navarra G. PB-106 

Nave K.A. PA-123 

Nawaz H. PB-27 

Nelander B. 62 

Nema N. 66 

Nemtseva E. PA-88 

Neri T. PA-22 

Neugebauer U. PB-28 

Neuhaus E.H. 22 

Ngolong E C. PA-89 

Niaura G. PA-94 

Nicoletti F.P. 13, PA-90 

Niebling S. PA-91

Nielsen O.F. 62 

Nikolic G.S. PA-83 

Noeske J. PA-2 

Nogaj B. PA-8, PA-11 

Notingher I. PA-114, PB-125 

Novikov E.G. 60 

Nowak C. PB-75, PB-83 

Nunes P. PB-42 

Nyitrai M. 44, PB-87 

Ochsenkühn M.A. PB-59 

Ofstad R. PB-107 

Ogilvie J.P. PA-13; PA-14 

Olasek M. PB-115 

Ollivier J. PB-31 

O’Neill S. PA-31 

Ortonne J.-P. 63 

Ortore M.G. PA-124 

Ostafin M. PA-11 

Oshtrakh M.I. 60 

Otero J.C. PA-77, PB-60 

Otto C. 74, PB-36, PB-120 

Ozel A.E. PB-70, PB-79 

Padan E. 6 

Padrão S. PA-29 

Pagnotta S.E. 39 

Palacky J. PA-84, PB-48, PB-51 

Palchykovska L. PA-111 

Pallavicini S. 80 

Pandelia M.E. PA-122, PB-82 

Pandey A. 66, PB-57 

Panne U. PA-104 

Pantusa M. PB-85 

Panzella L. 31, PA-60 

Panzica M. PA-92 

Paoletti S. PB-6 

Papazoglou E.S. PA-55 

Papiashvili N. 87 

Parker A.W. 92, PB-5 

Parkes G. 91 

Parola A.H. 87, PB-29 

Pärs M. PA-81 

Pascale F. PB-11 

Pascut F. PB-125 

Patel C. PA-55 

Patonay G. 88 

Paulsen H. 6 

Paus R. PA-73 

Pauwels E. PA-9 

Pazderka T. PB-102 

Pazzagli L. PB-112 

Pedzinski T. 93 

Perisic N. PB-107 

Perlik V. PA-16 

Perna G. PA-35 

Pessanha M. PA-116 

Peters J. 45, PA-18, PA-19 

Petersen W. PB-120 

Petrace H.I. 33 

Petri M. PB-90 

Petrone L. PA-93 

Pezzella A. PA-60 

Phillips R.S. PB-29 

xxi 

Picone G. PA-7 

Picone P. PB-96 

Pieridou G. 70 

Pijanka 91 

Pinzaru S. PB-12 

Piot O. 63, 90, PA-105 

Pizzanelli S. PA-4 

Plekan O. 41, PA-125 

Podstawka E. PA-94 

Pokkuluri P.R. PA-116 

Pol J. 65 

Polakovs M. PA-81 

Polverini E. PB-39, PB-84 

Polyanichko A. 95, PB-108 

Polyhach Y. 6 

Poon K.W.C. PB-30 

Popova A.D. PA-95 

Popova A.V. PB-109 

Popp J. 64, 86, PA-119, PB-13, 

PB-28, PB-116, PB-117 

Potara M. PA-25 

Povarova O.I. PB-62, PB-110 

Prati C. PB-24 

Praus P. PA-96 

Prince K.C. 41, PA-125 

Prisner T.F. PA-3 

Prochazka M. PA-68, PA-97 

Profant V. PA-98 

Proverbio E. PB-58 

Pully V.V. 74, PB-36 

Purchase R. 26 

Putsche M. PB-116 

Puustinin A. 42 

Pyshnyi D.V. 32 

Quartucci G. PA-35 

Quinn S.J. 92 

Quintes S. PA-123 

Raap J. 34 

Raccosta S. PA-41, PB-97, PB-111 

Raff J. PA-76 

Ragg N.C. PA-93 

Rak M. PA-123 

Rai B.K. PB-52 

Ramin M. PA-65 

Rasheed T. PA-20 

Rastogi V.K. 75, PA-20, PA-99, PB-52 

Raussens V. 58, PB-93, PB-101 

Ravikumar C. PA-59, PA-99 

Rayavarapu R.G. PB-120 

Reddy E. PA-31 

Redi C.A. PA-22 

Regulska E. PA-75, PA-100 

Reinicke M. PA-119 

Reis S. PB-56 

Remington S.J. 48 

Renciuk D. PB-46 

Ribeiro-Claro P.J.A. PA-103 

Richard C. PA-38 

Richter M. PB-117 

Richter R. 41, PA-125 

Ridderbusch O. 40 

Rifici S. PA-6

Rigler P. 78 

Rivasseau C. PB-31 

Rizzo T.R. 23, PA-87 

Rizzuti B. PB-85 

Robertazzi A. 101 

Rode J.E. PA-44 

Rodger A. 54 

Rodriguez-Casado A. PB-50 

Rodriguez Ortega PA-79 

M.P.G. 

Rodriguez Oviedo D. PB-2 

Rodriguez-Perez J.C. PA-101 

Roeffaers M.B.J. PB-122 

Roman M. PA-28 

Rondinelli C. 12 

Roosen-Runge F. PB-76 

Ropers M.H. PB-113 

Rösch P. PA-119, PB-116, PB-117 

Rosenkranz T. 78 

Rossi P.L. PB-24 

Roth A. PB-32 

Rouas G. PB-4 

Rousseau D.L. 42 

Roy A. 56, PB-99 

Ruano C. PA-77 

Ruckebusch C. 71 

Rudbeck M. 43 

Ruggerone P. 101 

Ruiz F. PA-24, PB-2 

Rusciano G. PB-36 

Ruysschaert J.M. 58, PA-89, PB-93, 

PB-101 

Ryu S.R. PA-40 

Saavedra Sagredo M.C. PB-63 

Sadlej J. PA-44 

Sadlo J. PB-49 

Saggu M. PA-122 

Sagstuen E. PA-9 

Sahu R.K. PA-86 

Sakmar T.P. PA-118, PB-63 

Salditt T. PA-123 

Salford L. PA-15 

Salgueiro C. PA-116 

Salman A. 77 

Salomo M. PB-124 

Salvi P.R. PA-47, PA-78 

Samoilova R.I. 34 

Samsonowicz M. PA-102 

San Biagio P.L. PB-96, PB-103, PB-111 

Sanchez-Cortés S. PA-42, PA-106, PB-10 

Sanda F. PA-16 

Sandt C. 91 

Sanson A. PB-7 

Santarella R. PB-105 

Santos C. PB-42 

Sardo M. PA-103 

Sarroukh R. 58 

Sasso A. PB-36 

Sauer M. 10 

Savorani F. PA-7 

Sbrana F. PB-112, PB-123 

Schäfer G. 30, PB-91 

xxii 

Schiffer M. PA-116 

Schirò G. PB-67 

Schlücker S. 84, PA-91 

Schmid P. PB-61 

Schmuck C.S. PA-91 

Schreiber F. PB-76 

Schreirer W.J. 94 

Schröder L. PB-5 

Schulte F. PA-104 

Schumacher W. PA-119 

Schumann C. 22, PA-45 

Schutz M. 84 

Schwalbe H. PA-2, PA-54 

Schweitzer-Stenner R. 53, PA-4, PA-21, PA-54 

Schwieger C. PB-113 

Sclafani M. PB-61 

Scorciapino M.A. 101 

Sebiskveradze D. PA-105 

Segundo A. PB-56 

Seckler R. PB-109 

Seidel S. PA-39 

Semeraro S. PB-6 

Semionkin V.A. 60 

Sergo V. PB-3, PB-6 

Sevilla P. PA-42, PA-106 

Seydel T. PB-76 

Sezer M. PA-107 

Shafranyosh I.I. PA-113 

Shakirova L. PB-18 

Sharma D. K. PA-20 

Sharma M. PB-52 

Sharrock P. PB-14 

Shastri S. 30 

Sheves M. 22 

Shibata M. 29 

Shnyrov V.L. PA-34 

Shu X. 48 

Shuvalov V.A. PA-117 

Skoda M.W.A. PB-76 

Sica F. PB-36 

Sicoli G. PB-45 

Silbey R.J. 26 

Silos I. PA-81 

Simakova P. PA-97 

Singh P. PA-63, PA-108, PA-109 

Singh R. PA-63, PA-109 

Sinibaldi R. PA-124 

Sionkowska A. 55, PB-104 

Sirlin C. PB-47 

Sironi L. 80 

Sirotkin S. PA-110 

Skopinska J. 55 

Sinyavsky N. PA-11 

Slobodyanyuk O. PA-62, PA-111 

Smulevich G. 13, PA-78, PA-90 

Sobolewski A.L. 15 

Sockalingum G.D. 73, 91 

Sotiriou C. PB-4 

Spilotros A. PA-112 

Spinozzi F. PA-124 

Sportelli L. PB-85, PB-98 

Spricigo R. PA-107

Squires A.M. PA-101 

Srivastav G. PA-63, PA-108, PA-109 

Srivastava A. PA-82 

Srivastava S.K. PA-91 

Staiano M. 57, PB-62, PB-110 

Staron K. PA-33 

Stavrov S.S. 28 

Stec H. PA-8 

Stefan R. 49 

Steinhoff H.J. PA-1 

Stepanek J. PA-46, PA-68, PA-96, 

PB-77 

Stepanenko Olesya 57, PB-62 

Stepanenko Olga V. PB-62, PB-95, PB-110 

Stirnal E. PA-2 

Stopin A. PB-7 

Strashenikov N. 87 

Strekowski L. 88 

Strohalm M. 65 

Stüber C. PB-116 

Subramaniam V. 74 

Sukhoviya M.I. PA-113 

Sulatskaya A.I. PB-110, PB-114 

Sulè-Suso J. 91 

Sundström V. PA-60 

Sureau F. PA-96, PB-69 

Surmacki J. 61, PB-33 

Susini J. PA-123 

Sweetenham C.S. PA-114 

Swislocka R. PA-75, PA-100, PA-115 

Szalontai B. PB-86 

Szatmari D. PB-87 

Szotek S. PB-20 

Szymborska-Malek K. PB-53 

Taddei P. PB-10, PB-24, PB-34 

Takekiyo T. 56 

Takenaka S. 93 

Talaya J. 75 

Tamburro A.M. PB-123 

Tandon P. PA-82 

Tardajos G. 75 

Tarpan M.A. PA-9 

Tatulian S.A. 25 

Techert S. PB-90 

Tehei M. PA-19 

Tekavec P.F. PA-13, PA-14 

Terazima M. 21 

Terentyeva T. PB-122 

Ter Halle A. PA-38 

Thompson M.K. PA-90 

Tiefenbach K.J. PB-94 

Tihomirova K. PB-19 

Tinti A. PB-10, PB-24, PB-35, 

PB-88, PB-106 

Tiribilli B. PB-112, PB-123 

Toderas F. PA-25 

Todorovic S. PA-67, PA-116 

Toffoletti A. 31 

Tokutomi S. 21 

Tomkinson J. PB-21 

Tominaga K. 29, PA-64 

Tommasini M. PB-8 

xxiii 

Toniolo C. 34 

Torreggiani A. PB-10, PB-35, PB-88, 

PB-106 

Towrie M. 92 

Trapp M. PA-19 

Trentmann O. 22 

Trimarchi A. PA-6 

Trofimov A.B. PA-125 

Trubetskaya O. PA-38 

Trubetskoj O. PA-38 

Trudeau T. 24 

Tsarfati I. PA-27 

Tsror L. 77 

Tsukada M. PB-34 

Tsvetkov Y.D. 34 

Tulkens P. PB-16 

Turoverov K.K. 57, PB-62, PB-95, 

PB-110, PB-114 

Turpin P.Y. PA-96 

Tutar Y. PB-54 

Tzaphlidou M. PA-71 

Ujfalusi Z. 44 

Untereiner V. 91 

Unruh T. PA-19 

Uritski A. PB-89 

Utesch T. PB-82 

Vallone B. 12 

Van Blitterswijk C.A. 74 

Vandenbussche G. PA-89 

Van Grondelle R. 47 

Van Pittius D.G. 91 

Van Etten J. PA-110 

Van Leeuwen T.G. PB-120 

Vaskivskyi L. PA-62, PA-111 

Vasi C. PB-72 

Vasilieva L.G. PA-117 

Vassalli M. PB-112, PB-123 

Vats J.K. 75, PB-52 

Vecoli M. PA-43 

Velcheva E.A. PA-95 

Velitchkova M. PA-23 

Verde C. 13 

Vergara A. PB-36 

Verhart N. 26 

Verhoefen M.-K. 30 

Verkhusha V.V. 57 

Versteegh G. PA-43 

Vidova V. 65 

Vitale A. PB-110 

Vitiur F. PB-6 

Vlassov V.V. 32 

Vogel R. PA-118, PB-63 

Vogel V. PB-22, PB-37 

Volker S. 26 

Volkov A. 6 

Vollmar M. PB-94 

Vollrath F. 54 

Volny M. 65 

Vorlickova M. PB-46 

Vorob’ev M.M. 96 

Vorobyev V. 95, PB-95 

Vos M.H. PB-81

Voué M. PB-16 

Vrabie V. PA-105 

Vrielinck H. PA-9 

Vulpiani A. 100 

Wachtveitl J. 30, PB-44 

Wagner C. PB-124 

Walfisch S. PA-86 

Walter A. PA-119 

Walter K. PB-90 

Wanderlingh U. PA-6, PB-72 

Wang D. PB-38 

Wang R. PA-57 

Waroquier M. PA-9 

Wassef M. PA-74, PB-11, PB-15, 

PB-25 

Weber I. 30 

Weidinger I.M. PA-107 

Wharton C.W. PA-120 

Wich P.C. PA-91 

Wielgus-Kutrowska B. PA-33, PB-115 

Wieser H. PB-108 

Wikstrom M. 42 

Wille G. PB-91 

Wisitruangsakul N. PA-61, PB-82 

Wittinghofer A. PA-1 

Wöhnert J. PA-2 

Wojdyla M. 92 

Wolf M. PA-45 

Wolf M.M.N. PA-121 

Wollenberger U. PA-107 

Wood B.R. PA-73 

Wrobel T. PA-10 

Wu L. 40, 56 

Wysokinski R. PB-23 

Yabushita A. 69 

Yadav R. A. PA-63, PA-108, PA-109, 

PB-40 

Yadav T.K. PB-40 

Yang Y. 91 

Ye S. PA-118 

Yeh S.-R. 42, PB-92 

Yesylevskyy S. PB-78 

Zaitseva E. PB-63 

Zalloum W.A. 32 

Zandomeneghi G. PA-4 

Zanoni M. PA-22 

Zaytseva I.L. PA-125 

Zebger I. PA-61, PA-122, PB-82 

Zenkova M.A. 32 

Zerbi G. PB-8 

Zhang D. 83 

Zhang F. PB-76 

Zhang W. 41 

Zhorov E. PB-38 

Zhu J. PB-83 

Zienicke B. PA-45 

Zikmanis P. PB-18 

Zinth W. 94 

Zoladek A. PB-125 

Zorn S. PB-76 

Zuccotti M. PA-22 

xxiv

XIII European Conference on the Spectroscopy of ... - ecsbm 2009

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