Plenarvorträge - DPG-Tagungen

Symposium Life Sciences on the Nanometer Scale - Physics Meets Biology Mittwoch
In very dilute paramagnets selective dynamic nuclear polarisation of
proton spins close to paramagnetic centres provides an amplitude of polarised
neutron scattering which is considerably stronger than that of
magnetic neutron scattering. This is shown for bovine liver catalase,
which is a homotetramer of 506 amino acids (MW=230 kD) with tyrosin-
369 and possibly other tyrosins in a radical state. From comparison of
time-resolved polarised neutron small-angle scattering with simultaneous
NMR measurements it appears that at the onset of dynamic nuclear
polarisation a large majority of the polarised protons are close to the
unpaired electron of the tyrosyl radicals. Polarised proton spin domains
are built up in less than 10 s, while the polarisation of the bulk proton
remains low. Comparison of the time-resolved neutron scattering data
with model calculations confirms the existence of tyrosyl-369
SYLS 3.56 Mi 16:00 B
Plasmon-enhanced Ramanspectroscopy in the Near-field of
Dynamically Tuneable Nanostructured Gratings — •Dominic
Zerulla, Gereon Isfort, Frank Katzenberg, Micha Kölbach,
and Klaus Schierbaum — Heinrich-Heine Universität Düsseldorf,
IPkM, AG Physikalische Methoden für Biologie und Medizin,
Universitätsstr. 1, D-40225 Düsseldorf
Ramanspectroscopy has proven to be a powerful tool in the investigation
of biological molecules. However, additional enhancements are often
needed. As a first enhancement we use the resonance effect by tuning the
laser wavelength onto a certain electronic excitation.
Apart from Surface Enhanced Raman Scattering (SERS) as a second
enhancement technique which is ill-suited to the problem, one solution
to the problem is given by exciting a surface-plasmon-wave on a surface
which is specifically tailored to the system. Confining the enhancement
to its electromagnetic part by means of smooth surfaces or a regular
metallic grating leads to predictable electromagnetic field strengths with
decay lengths of about 100 nm. In order to meet the requirements of a
specific plasmon excitation and the resonance conditions simultaneously,
it is extremely helpful to tune the grating properties. This is done by
using specific gratings which consist of quantum wire-like structures of
metals on a polymer base whose spacings can be changed dynamically
from 0 nm to several hundredth of nm. Such systems can be optimized to
yield high sensitivity and selectivity along with decay length appropriate
for detection of macromolecular mechanisms at membranes.
SYLS 3.57 Mi 16:00 B
Investigation of the range of electromagnetic Raman-
Enhancement in biological Films by means of SAMs —
•Gereon Isfort, Micha Kölbach, Dominic Zerulla, and
Klaus Schierbaum — Heinrich-Heine-Universität Düsseldorf, IPkM,
Materialwissenschaften, AG Physikalische Methoden für Biologie und
Medizin, Universitätsstr. 1, D-40225 Düsseldorf, Germany
Raman-Spectroscopy is a powerful tool for probing the structure and
conformation of proteins. Biological environments produce a large number
of signals, not easily to assign. A selected enhancement might help
to exclude the environmental signals.
The ATR-SPP (Attenuated Total Reflection - Surface Plasmon-
Polariton) technique enhances the electromagnetic field at thin metal
interfaces. The exponential decrease of the evanescent field confines the
range of the enhancement. The Raman activity of the molecules inside
this field is strongly accentuated compared to the molecules outside. To
prove this theoretically trivial statement under real conditions (not perfect
plane metallic layer, unclear microscopic dielectric constants) and to
get quantitative results of the decay lengths, we have made a systematic
approach by using specific self-assembled monolayers of definitive thickness
as spacers in order to vary the distance between the metallic layer
and the Raman-active sample deposited on the SAMs.
SYLS 3.58 Mi 16:00 B
Evaluation of Laser Scanning Microscopic Methods on
Biological Molecules in Membranes — •Micha Kölbach,
Dominic Zerulla, Kerstin Elfrink, Gereon Isfort, and
Klaus Schierbaum — Heinrich-Heine-Universität Düsseldorf, IPkM,
Materialwissenschaften, AG Physikalische Methoden für Biologie und
Medizin, Universitätsstr. 1, D-40225 Düsseldorf, Germany
As of today the infection mechanisms of the prion proteins are still not
completely understood.
In order to investigate these infectious biological molecules in membranes,
we have tested a multitude of different microscopic approaches, basing on
fluorescence as well as Raman spectroscopy. We have decided to pursue
this goal by building two different high sensitive microscopes for low light
detection.
The first one uses only reflecting light and offers, through the use of a
high precision xyz micropositioning table, a scanning mode. This system
is able to supply a large quantity of information from each single sample
by providing a full spectrum for each scanned point. Since the scanning
mode is a long lasting process, the second microscope makes use of a multichannel
detector in conjunction with dispersive components or optical
filters, and therefore offers a faster recording of fluorescence or Raman
images. It also features the use of both reflecting light and see-through
mode. In connection with the use of photoncounting equipment we strive
to detect single molecule fluorescence of labelled Acetylcholinesterase
molecules bound via GPI-anchors in a lipid bilayer, a system already
close to prion proteins in the same membrane.
SYLS 4 Symposium ”Life Sciences on the Nanometer Scale - Physics Meets Biology”
Zeit: Donnerstag 09:30–11:00 Raum: H 37
Hauptvortrag SYLS 4.1 Do 09:30 H 37
Single Molecule Mechanics of Cytoskeletal Proteins —
•Matthias Rief — Lehrstuhl fuer Biophysik E22 der TU Muenchen,
James-Franck-Str., 85748 Garching
The mechanical properties of cytoskeletal proteins and molecular motors
are important for their function in vivo. However, this information
has become accessible only recently through the invention of single
molecule techniques like atomic force microscopy. We have used AFM
based force spectroscopy to investigate the mechanical response of the
coiled-coil domains of myosin II and the actin cross-linking protein Ddfilamin.
We find that the myosin coiled-coil is a highly elastic protein structure
that undergoes an unfolding/refolding transition at 25 pN. Unlike
all other proteins investigated so far this transition occurs in equilibrium.
These measurements show that a coiled-cloil is able to produce
forces during folding. Ddfilamin is an actin crosslinking protein from dictyostelium
discoideum. Using single molecule unfolding experiments we
show that one of the immunoglobulin domains of this protein unfolds at
low forces via a stable intermediate. We have used amino-acid inserts into
the loops of this domain to map the structure of this intermediate. We
show evidence that the intermediate is also populated during folding of
this domain which increases the refolding rates drastically. Low unfolding
forces together with fast refolding kinetics suggest an in-vivo role for this
domain as a reversibly extensible element under mechanical strain.
SYLS 4.2 Do 10:00 H 37
Atomic Force Microscopy and Spectroscopy of Specific Protein-
DNA Interaction — •F. Bartels 1 , B. Baumgarth 2 , A. Becker 2 ,
R. Ros 1 , and D. Anselmetti 1 — 1 Experimental Biophysics, Faculty of
Physics, Bielefeld University — 2 Genetics, Faculty of Biology, Bielefeld
University
Specific protein-DNA interaction is fundamental for all aspects of gene
expression. In the soil bacterium Sinorhizobium meliloti 2011, the protein
ExpG controls the biosynthesis of polysaccharide polymers, which
promote the bacterium‘s symbiosis with alfalfa plants for means of fixing
molecular nitrogen. We investigated the molecular mechanism of
binding of ExpG to three associated DNA target sequences both with
standard biochemical methods and single molecule force spectroscopy
based on the atomic force microscope (AFM). AFM imaging was used
in addition to obtain topographical information regarding the process of
binding. We demonstrated binding in a sequence specific manner, with
unbinding forces ranging from 50 to 165 pN in a logarithmic dependence
from the loading rates of 70 to 79,000 pN/s. Two different regimes