Content
2007
Metal-Enhanced Fluorescence of Chlorophylls in Single Light-Harvesting Complexes
22.12.2007
Ensemble and single-molecule spectroscopy demonstrates that both emission and absorption of peridinin-chlorophyll-protein photosynthetic antennae can be largely enhanced through plasmonic interactions. We find up to 18-fold increase of the chlorophyll fluorescence for complexes placed near a silver metal layer. This enhancement, which leaves no measurable effects on the protein structure, is observed when exciting either chlorophyll or carotenoid and is attributed predominantly to an increase of the excitation rate in the antenna. The enhancement mechanism comes from plasmon-induced amplification of electromagnetic fields inside the complex. This result is an important step toward applying plasmonic nanostructures for controlling the optical response of complex biomolecules and improving the design and functioning of artificial light-harvesting systems.
Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations
18.12.2007
PNAS,
2007,
doi: 10.1073/pnas.0705458104,
vol. 104 no. 51 20268-20273,
published on 18.12.2007
PNAS, online article
PNAS, online article
Nanomanipulation of biomolecules by using single-molecule methods and computer simulations has made it possible to visualize the energy landscape of biomolecules and the structures that are sampled during the folding process. We use simulations and single-molecule force spectroscopy to map the complex energy landscape of GFP that is used as a marker in cell biology and biotechnology. By engineering internal disulfide bonds at selected positions in the GFP structure, mechanical unfolding routes are precisely controlled, thus allowing us to infer features of the energy landscape of the wild-type GFP. To elucidate the structures of the unfolding pathways and reveal the multiple unfolding routes, the experimental results are complemented with simulations of a self-organized polymer (SOP) model of GFP. The SOP representation of proteins, which is a coarse-grained description of biomolecules, allows us to perform forced-induced simulations at loading rates and time scales that closely match those used in atomic force microscopy experiments. By using the combined approach, we show that forced unfolding of GFP involves a bifurcation in the pathways to the stretched state. After detachment of an N-terminal alpha-helix, unfolding proceeds along two distinct pathways. In the dominant pathway, unfolding starts from the detachment of the primary N-terminal beta-strand, while in the minor pathway rupture of the last, C-terminal beta-strand initiates the unfolding process. The combined approach has allowed us to map the features of the complex energy landscape of GFP including a characterization of the structures, albeit at a coarse-grained level, of the three metastable intermediates.
Visualizing single-molecule diffusion in mesoporous materials
29.11.2007
Periodic mesoporous materials formed through the cooperative
self-assembly of surfactants and framework building blocks can
assume a variety of structures1–3, and their widely tuneable properties
make them attractive hosts for numerous applications4–7.
Because the molecular movement in the pore system is the most
important and defining characteristic of porous materials8, it is of
interest to learn about this behaviour as a function of local structure.
Generally, individual fluorescent dye molecules can be used
as molecular beacons with which to explore the structure of—and
the dynamics within—these porous hosts, and single-molecule fluorescence techniques provide detailed insights into the dynamics of various processes, ranging from biology to hetero- geneous catalysis16. However, optical microscopy methods cannot directly image the mesoporous structure of the host system accommodating the diffusing molecules, whereas transmission electron microscopy provides detailed images of the porous structure, but no dynamic information. It has therefore not been possible to ‘see’ how molecules diffuse in a real nanoscale pore structure. Here we present a combination of electron microscopic mapping and optical single-molecule tracking experiments to reveal how a single luminescent dye molecule travels through linear or strongly curved sections of a mesoporous channel system. In our approach we directly correlate porous structures detected by transmission electron microscopy with the diffusion dynamics of single molecules detected by optical microscopy. This opens up new ways of understanding the interactions of host and guest.
Relative binding affinities of chlorophylls in peridinin–chlorophyll–protein reconstituted with heterochlorophyllous mixtures
25.10.2007
Peridinin–chlorophyll–protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and singlemolecule spectroscopy. They can be obtained by reconstituting
the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these ‘‘half-mers’’, homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium
carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.
Light-triggered beta-hairpin folding and unfolding
02.10.2007
A light-switchable peptide is transformed with ultrashort pulses
from aß-hairpin to an unfolded hydrophobic cluster and vice versa.
The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining
density functional theory with molecular mechanics is used to
interpret the absorption transients. Illumination of the b-hairpin
state triggers an unfolding reaction that visits several intermediates
and reaches the unfolded state within a few nanoseconds. In
this unfolding reaction to the equilibrium hydrophobic cluster
conformation, the system does not meet significant barriers on the
free-energy surface. The reverse folding process takes much longer
because it occurs on the time scale of 30 micro s The folded state has
a defined structure, and its formation requires an extended search
for the correct hydrogen-bond pattern of the ß-strand.
Direct Observation of Active Protein Folding Using Lock-in Force Spectroscopy
17.08.2007
Direct observation of the folding of a single polypeptide chain can provide important information about the thermodynamic states populated along its folding pathway. In this study we present a lock-in force spectroscopy technique that improves resolution of AFM force spectroscopy to 400 fN. Using this technique we show that immunoglobulin domain 4 from dictyostelium discoideum filamin (ddFLN4) refolds against forces of approx. 4 pN. Our data show folding of this domain proceeds directly from an extended state and no thermodynamically distinct collapsed state of the polypeptide prior to folding is populated. Folding of ddFLN4 under load proceeds via an intermediate state. Three-state folding allows ddFLN4 to fold against significantly larger forces than would be possible for a mere two-state folder. We present a general model for protein folding kinetics under load that can predict refolding forces based on chain length and zero force refolding rate.
Infrared Studies of Small Azobenzene Peptides: Unexpectedly Slow Reactions on the Time Range of Minutes
10.08.2007
J. Phys. Chem. B,
2007,
111,
10481-86
published on 10.08.2007
The Journal of Physical Chemistry B, online article
The Journal of Physical Chemistry B, online article
Infrared absorption experiments on light-triggered azobenzene peptides have been performed below and above the freezing point of the solvent dimethyl sulfoxide (DMSO). Even 20 K below the freezing point, illumination of the azobenzene chromophore resulted in IR absorption changes indicative of light-induced structural rearrangements of the peptide. In addition, new conformational states could be found at low temperature, which involve the formation of additional hydrogen bonds. In one sample the new low-temperature state survived melting and reheating and disappeared at 298 K only on the time scale of 10 min. The observations indicate that at low temperatures the peptide, together with traces of water present in the sample, forms a shell in the vicinity of the chromophore that facilitates internal motion even in the rigid cage of frozen DMSO.
Photochemical Z to E Isomerization of a Hemithioindigo/Hemistilbene omega-Amino Acid
05.07.2007
The molecule HTI, which combines hemithioindigo and hemistilbene molecular parts, allows reversible switching between two isomeric states. Photochromic behaviour of the HTI molecule is observed by irradiation with UV/Vis light. The photochemical reaction, a Z/E isomerization around the central double bond connecting the two molecular parts, is investigated by transient absorption and emission spectroscopy. For a special HTI molecule, namely, an omega-amino acid, the Z!E isomerization process occurs on a timescale of 30 ps. In the course of the reaction fast processes on the 1–10 ps timescale are observed which point to motions of the molecule on the potential-energy surface of the excited state. The combination of transient absorption experiments in the visible spectral range with time-resolved fluorescence and infrared measurements reveal a photochemical pathway with three intermediate states. Together with a theoretical modelling procedure the experiments point to a sequential reaction scheme and give indications of the nature of the involved intermediates.
Comparing a Photoinduced Pericyclic Ring Opening and Closure: Differences in the Excited State Pathways
14.06.2007
Journal of the American Chemical Society,
2007,
129,
8577-84
published on 14.06.2007
Journal of the American Chemical Society, online article
Journal of the American Chemical Society, online article
The photochromicity of fulgimides rests on the existence of open (E) and closed ring (C) isomers. As predicted by the Woodward-Hoffmann rules both isomers can photochemically be interconverted. This interconversion has been studied by femtosecond fluorescence and transient absorption spectroscopy.
For either direction (E toC cyclization and C to E cycloreversion) a biphasic fluorescence decay on the 0.1-1 ps time scale is observed. The longer time constants of the decays equal the formation times of the photoproducts. The time constants retrieved (0.06 and 0.4 ps for E toC, 0.09 and 2.4 ps for C to E) and the associated spectral signatures differ substantially. This indicates that no common excited-state pathway for the two directions exists, as one would infer from a simple Woodward-Hoffmann consideration. These findings support recent quantum dynamic calculations on the excited-state topology of fulgimides.
Dem Molekül auf der Spur
01.06.2007
Auf welchem Weg dringt ein Virus bei einer Infektion
in eine Zelle ein? Wie bewegen sich Motorproteine in
Zellen oder einzelne Moleküle in nanoporösen Materialien?
Das Verfolgen (Tracking) einzelner Teilchen unter
dem Fluoreszenzmikroskop erlaubt es, diese und
ähnliche Fragen aus der Biophysik, der Medizin und
den Materialwissenschaften zu beantworten.
Solarzellen nach biologischem Vorbild?
29.04.2008
Physik in unserer Zeit,
2008,
39,
113-4
published on 29.04.2007
Physik in unserer Zeit, online article
Physik in unserer Zeit, online article
Forscher der LMU München haben gezeigt, dass sich mit Hilfe von Silbernanoteilchen die Effizienz von Lichtsammelkomplexen enorm steigern lässt. Da es sich hierbei um die zentralen Funktionsträger für das Einsammeln von Licht bei der Photosynthese handelt, könnte diese Entdeckung bedeutsam für die Entwicklung neuartiger Hybrid-Solarzellen sein.
Femtosecond stimulated Raman microscopy
12.04.2007
Appl. Phys. B,
2007,
87,
389-93
published on 12.04.2007
Applied Physics B Lasers and Optics, online article
Applied Physics B Lasers and Optics, online article
A novel type of non-linear Raman microscopy, femtosecond stimulated Raman microscopy (FSRM), is introduced. It employs femtosecond white light pulses and intense picosecond pulses which are derived from a femtosecond laser/amplifier system. The pulses are coupled into a microscope set-up and induce a stimulated Raman process at the focus. The Raman interaction spectrally modulates the white light. These modulations are read-out in multi-channel fashion and allow recording of a complete Raman spectrum of the focal region. By raster-scanning the sample, complete Raman images can be obtained. Raman images of polystyrene beads in water demonstrate the feasibility of the approach.
Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage Phi29 Capsids
20.02.2007
The bacteriophage Phi29 uses a molecular motor to drive its genome into a preformed protein capsid. The central part of this molecular motor is formed by a ring of twelve proteins called the connector. Symmetry and structural arguments have let to the so called rotation hypothesis, i.e. a rotation of the connector is used to drive the DNA into the capsid, which for the last 30 years has been the most prominent model for the function of this motor. We have tested this hypothesis using single-molecule fluorescence polarisation and could show, that the connector does not rotate during packaging. Therefore a new model for motor function had to be developed.
This manuscript was highlighted by the editorial team of PLOS Biology in this Commentary:
Does Bacteriophage Phi29 Package its DNA with a twist?
