Plenarvorträge - DPG-Tagungen

Symposium Life Sciences on the Nanometer Scale - Physics Meets Biology Mittwoch
ing of proteins in the absorption range of their aromatic amino acids.
Tyrosine is most effective as a hole burning probe most probably due
to light-induced hydrogen abstraction at the hydroxy-group. We applied
the technique to BPTI, insuline and ribonuclease. We performed Starkeffect
and pressure tuning experiments with the holes to shed light on
the electrostatic and elastic properties of the proteins.
SYLS 3.8 Mi 16:00 B
Spectal diffusion experiment with a denatured protein
— •Vladimir Ponkratov and Josef Friedrich — Physik-
Department E14, Lehrstuhl fuer Physik Weihenstephan, TU Muenchen
Spectral diffusion broadening of a persistent spectral hole burnt into
the absorption of a cytochrome c-type protein in its unfolded state is investigated
and compared to the corresponding broadening in the native
state. Spectral diffusion broadening is much larger in the unfolded state.
We found that the time law which governs spectral diffusion changes
from a power law to a seemingly logarithmic law as the aging time of the
protein encreases.
SYLS 3.9 Mi 16:00 B
Direct Observation of Tiers in the Energy Landscape of a Chromoprotein:
A Single-Molecule Study — •Martin Richter 1 ,
C. Hofmann 1 , T.J. Aartsma 2 , H. Michel 3 , and J. Köhler 1 —
1 Experimental Physics IV, University of Bayreuth — 2 Department of
Biophysics, Leiden University — 3 Department of Molecular Membrane
Biology, Max-Planck Institute of Biophysics, Frankfurt
We report the observation of spectral fluctuations in the B800 band of
light harvesting 2 (LH2) complexes from Rhodospirillium molischianum.
The electronically excited states of these chromophores are mainly localized
on individual BChl a molecules and can serve as local probes to
monitor changes in the protein envirornment. The data provide information
about the organization of the energy landscape of the protein in
tiers that can be characterized by an average barrier height. In addition
a clear correlation for the transition rates between those states and the
energy separation of the levels involved is uncovered.
SYLS 3.10 Mi 16:00 B
High-Resolution Near-Field Fluorescence Microscopy of Single
Nuclear Pore Complexes in an Intact Nuclear Envelope — •C.
Höppener 1 , J.-P. Siebrasse 2 , U. Kubitscheck 2 , R. Peters 2 ,
H. Fuchs 1 , and A. Naber 3 — 1 Physikalisches Institut, Westfälische
Wilhelms-Universität, 48149 Münster — 2 Institut für Medizinische
Physik und Biophysik, Westfälische Wilhelms-Universität, 48149
Münster — 3 Institut für Angewandte Physik, Universität Karlsruhe
(TH), 76131 Karlsruhe
The nuclear pore complex (NPC) is a large macromolecular protein
assembly embedded in the nuclear envelope (NE) of a eukaryotic cell
and mediates the highly selective, bi-directional exchange of all kinds of
molecules between cytoplasm and nucleus. So far single NPCs could not
be resolved by optical means since the NPCs are densely packed in the
membrane. We present high-resolution near-field optical images of a fluorescently
labelled NE [1] performed in a buffer solution. These images
represent the first example of a high-resolution scanning near-field optical
measurement of a functionally intact biomembrane. A super-resolution
of 60nm enables us to optically identify for the first time single, closely
neighboured NPCs. Furthermore, the appearance of the NPCs markedly
varies with the primary antibody used for fluorescence labelling, thus revealing
information about the location of a specific nucleoporin within
the NPC.
[1] C. Höppener, D. Molenda, H. Fuchs, and A. Naber, J. Microsc. 210,
288 (2003)
SYLS 3.11 Mi 16:00 B
Time-resolved Fluorescence Resonance Energy Transfer on
DNA duplexes — •Petra Müller, Jürgen Köhler, and Dagmar
Klostermeier — Experimental Physics IV, University of Bayreuth,
Bayreuth
Fluorescence resonance energy transfer (FRET) is an established technique
to determine intramolecular distances between 1 and 10 nm. In
time-resolved FRET experiments, inter-fluorophore distance information
is retrieved from the analysis of fluorescence emission decays of the donor
fluorophore in the absence and presence of the acceptor fluorophore.
Here we report distance measurements using our home-built timeresolved
FRET setup. Excitation is performed using the frequencydoubled
output of a pulsed titanium:sapphire laser, and the nanosecond
decay profile of the donor is measured via time-correlated single photon
counting. Calibration with a series of donor-acceptor labelled DNA
molecules yields inter-fluorophore distances in good agreement with calculated
values based on standard DNA B-form geometry and the chemistry
of fluorophore attachment. Furthermore, multiple distance distributions
for various mixtures of DNA molecules of different lengths can be
extracted correctly from the donor decays.
Having established the possibilities and limitations of our time-resolved
FRET set-up we will now employ this technique to identify functional
conformers of proteins that modulate nucleic acid structures, such as
helicases and topoisomerases.
SYLS 3.12 Mi 16:00 B
A fluorescence anisotropy-based activity assay for the RNA-
Helicase DbpA — •Niklas Nachtmann and Dagmar Klostermeier
— Experimental Physics IV, University of Bayreuth, Bayreuth
The Escherichia coli protein DbpA is an RNA-helicase involved in ribosome
biogenesis. It comprises conserved helicase motifs, such as a Walker
A motif and a DEAD box involved in ATP binding and hydrolysis, an
arginine-rich motif that mediates RNA binding, and a SAT motif responsible
for coupling of ATPase and RNA unwinding activities.
We have developed a DbpA helicase activity assay using a fluoresceinlabeled
model substrate and fluorescence anisotropy. The fluorescently
labeled substrate binds to DbpA with high affinity, and the unwinding
of its short double-stranded region can be followed via a concomitant
decrease in fluorescein anisotropy. This anisotropy decrease is only observed
in the presence of ATP, not ADP, consistent with ATP-dependent
helix unwinding. Furthermore, a mutant of DbpA in which the conserved
SAT motif has been converted to AAA does not exhibit helicase activity
towards this substrate, as monitored by fluorescence anisotropy.
Despite minor sequence differences in the corresponding ribosomal
RNA region, this activity test is applicable to the homologous RNA helicase
YxiN from Bacillus subtilis. This anisotropy-based activity test
will be an invaluable means to assay helicase constructs manipulated for
single molecule FRET experiments for wild-type like activity.
SYLS 3.13 Mi 16:00 B
Multivariate Statistical Analysis applied to Single-Molecule
Spectra — •Jürgen Baier 1 , C. Hofmann 1 , M. Richter 1 ,
M. Schatz 2 , H. Michel 3 , M. van Heel 4 , and J. Köhler 1 —
1 Experimental Physics IV, University of Bayreuth — 2 Image Science
Software GmbH, Berlin — 3 Department of Membrane Biology, MPI of
Biophysics, Frankfurt — 4 Department of Biological Sciences, Imperial
College, London
The spectral width of an optical transition from an ensemble of
molecules reflects the statistical distribution of local environments and
is commonly termed inhomogeneous linewidth. As is well known, singlemolecule
spectroscopy allows to supass this phenomenon and to obtain
the homogeneous linewidth of the optical transition. However, the observed
linewidth corresponds to the temporal average and the above mentioned
argument holds true only if the experimental observation time for
the single-molecule transition is short with respect to the timescale of
the fluctuations in the sample.
Here we demonstrate that the combination of fast data acquisition
and pattern recognition algorithms which are ususally employed in cryoelectron
microscopy allows us to elucidate further information from
single-molecule lineshapes if this perequisite is not fulfilled.
SYLS 3.14 Mi 16:00 B
Optimizing Water-Soluble Quantum Dots for Biological Application
— •Vladimir V. Breus 1 , Colin D. Heyes 1 , Andrei Yu.
Kobitski 1 , Kirill V. Anikin 1 , and G. Ulrich Nienhaus 1,2 —
1 Department of Biophysics, University of Ulm, D - 89069 Ulm, Germany
— 2 Department of Physics, University of Illinois Urbana-Champaign,
Urbana, IL 61801, USA
The use of quantum dots has several advantages over fluorescent dyes
for biological application. They are bright, easy tunable in color, and
extremely stable against photo bleaching (> 10 8 emitted photons). Various
bifunctional ligands were used to obtain water-soluble, biocompatible
ZnS coated CdSe quantum dots. The chemical stability, photoluminescence
efficiency and fluorescent blinking of the different samples were
compared. The optimized water-soluble quantum dots were used in longtimescale
single-molecule imaging of lipid diffusion in membranes.