Silke Oellerich

Refinement of the x-ray structure of the RC-LH1 core complex from Rhodopseudomonas palustris by single-molecule spectroscopy

A unique combination of single-molecule spectroscopy with numerical simulations has allowed us to achieve a refined structural model for the bacteriochlorophyll a (BChl a) pigment arrangement in reaction center–light-harvesting 1 core complexes of Rhodopseudomonas palustris. Details in the optical spectra, such as spectral separation and mutual polarizations of spectral bands, are compared with results from numerical simulations for various models of the BChl a arrangement that were all well within the 4.8-Å limit of the accuracy of the available x-ray structure. The experimental data are consistent with a geometry where 15 BChl a dimers, each taken homologous to those from light-harvesting 2 complex from Rhodopseudomonas acidophila, are arranged in an overall elliptical structure featuring a gap on the long side of the ellipse.

Conformational equilibria and dynamics of cytochrome c induced by binding of sodium dodecyl sulfate monomers and micelles

Circular dichroism, nuclear magnetic resonance, electron paramagnetic resonance, UV-vis absorption, and resonance Raman (RR) spectroscopic techniques were employed to study protein and heme structural changes of cytochrome c (Cyt-c) induced by sodium dodecyl sulfate (SDS) monomers and micelles via hydrophobic and electrostatic interactions, respectively. Both modes of interactions cause the transition to the conformational state B2, which is implicated to be involved in the physiological processes of Cyt-c. At sub-micellar concentrations of SDS, specific binding of only ca. three SDS monomers, which is likely to occur at the hydrophobic peptide segment 81–85, is sufficient for a complete conversion to a B2 state in which Met80 is replaced by His33 (His26). These heme pocket structural changes are not linked to secondary structure changes of the protein brought about by nonspecific binding of SDS monomers in different regions of the protein. Upon binding of micelles, B2 high-spin species can also be stabilized by electrostatic interactions. In addition, the micelle interaction domain is located on the front surface of Cyt-c, which includes a ring-like arrangement of lysine residues appropriate for binding one micelle. According to freeze-quench RR and stopped-flow experiments, state B2 is formed on the long millisecond timescale and reveals a complex dependence on the SDS concentration that can be interpreted in terms of competitive binding of monomers and micelles.

Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX

Low-temperature (1.4 K), single-molecule fluorescence-excitation spectra have been recorded for individual reaction center–light-harvesting 1 complexes from Rhodopseudomonas palustris and the PufX− strain of Rhodobacter sphaeroides. More than 80% of the complexes from Rb. sphaeroides show only broad absorption bands, whereas nearly all of the complexes from Rps. palustris also have a narrow line at the low-energy end of their spectrum. We describe how the presence of this narrow feature indicates the presence of a gap in the electronic structure of the light-harvesting 1 complex from Rps. palustris, which provides strong support for the physical gap that was previously modeled in its x-ray crystal structure.

The Electronically Excited States of LH2 Complexes from Rhodopseudomonas acidophila Strain 10050 Studied by Time-Resolved Spectroscopy and Dynamic Monte Carlo Simulations. I. Isolated, Non-Interacting LH2 Complexes

We have employed time-resolved spectroscopy on the picosecond time scale in combination with dynamic Monte Carlo simulations to investigate the photophysical properties of light-harvesting 2 (LH2) complexes from the purple photosynthetic bacterium Rhodopseudomonas acidophila. The variations of the fluorescence transients were studied as a function of the excitation fluence, the repetition rate of the excitation and the sample preparation conditions. Here we present the results obtained on detergent solubilized LH2 complexes, i.e., avoiding intercomplex interactions, and show that a simple four-state model is sufficient to grasp the experimental observations quantitatively without the need for any free parameters. This approach allows us to obtain a quantitative measure for the singlet-triplet annihilation rate in isolated, noninteracting LH2 complexes.

The Electronically Excited States of LH2 Complexes from Rhodopseudomonas acidophila Strain 10050 Studied by Time-Resolved Spectroscopy and Dynamic Monte Carlo Simulations. II. Homo-Arrays Of LH2 Complexes Reconstituted Into Phospholipid Model Membranes

We performed time-resolved spectroscopy on homoarrays of LH2 complexes from the photosynthetic purple bacterium Rhodopseudomonas acidophila. Variations of the fluorescence transients were monitored as a function of the excitation fluence and the repetition rate of the excitation. These parameters are directly related to the excitation density within the array and to the number of LH2 complexes that still carry a triplet state prior to the next excitation. Comparison of the experimental observations with results from dynamic Monte Carlo simulations for a model cluster of LH2 complexes yields qualitative agreement without the need for any free parameter and reveals the mutual relationship between energy transfer and annihilation processes.

Spectral Diffusion and Electron-Phonon Coupling of the B800 BChl a Molecules in LH2 Complexes from Three Different Species of Purple Bacteria

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

Low-temperature single-molecule spectroscopy on photosynthetic pigment–protein complexes from purple bacteria

The primary reactions of purple bacterial photosynthesis take place within two well characterized pigment–protein complexes, the core Reaction Center–Light Harvesting 1 (RC–LH1) complex and the more peripheral Light Harvesting 2 (LH2) complex. These antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction-centers, where it is used to ‘power’ the photosynthetic redox reaction. This review provides an overview of how the character of the electronically excited states of these pigment–protein complexes are determined by quantum mechanics and how the respective spectral signatures can be observed by single-molecule spectroscopy.

Freeze-Quench Resonance Raman and Electron Paramagnetic Resonance Spectroscopy for Studying Enzyme Kinetics: Application to Azide Binding to Myoglobin

In the present work we have developed a reliable approach for probing the reaction dynamics of metalloproteins on the millisecond time scale. It is based on the combination of the freeze-quench method with resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopy. The reactions are initiated in a mixing chamber and rapidly quenched at low temperature in liquid isopentane after variable delay times. The experimental device is designed in such a way that the same frozen samples can be subsequently studied by two analytical techniques, thereby providing complementary information about the active site structures of intermediate states of the enzyme. In particular, the present setup permits the measurement of high-quality RR spectra despite the interference by the Raman bands of isopentane. With the use of the azide binding reaction to myoglobin as a test case, it is found that RR spectroscopy allows a reliable determination of rate constants, whereas the quantitative analysis of the EPR spectra is associated with a relatively high and unavoidable uncertainty mainly due to irreproducible packing of the freeze-quenched samples.

AFM characterization of spin-coated multilayered dry lipid films prepared from aqueous vesicle suspensions.

We present a detailed AFM study on multilayered dry lipid films prepared from aqueous vesicle suspensions. Different preparation techniques were applied in order to optimize the preparation of homogeneous lipid films of various film thicknesses. Suspensions of preformed DOPC/DPPC vesicles were adsorbed onto indium tin oxide-coated glass coverslips, a substrate also commonly employed for the formation of giant liposomes. We found that the homogeneity of the lipid films could substantially be improved when applying a spin-coating step during the film preparation. These films were much more homogeneous than those prepared by conventional drop-casting and in addition the film thickness could be controlled. When using a combination of vesicle adsorption and spin-coating the quality and thickness of the films depended crucially on the lipid concentration of the vesicle suspension, the adsorption temperature and the adsorption time. For lipid films prepared by direct spin-coating the lipid concentration and the applied spin-coating sequence were critical parameters for the quality and thickness of the deposited lipid films.

Simultaneous wide-field imaging and spectroscopy of localized fluorophores.

A method that combines fluorescence imaging and spectroscopy of single molecules at room temperature is presented. This approach allows us to identify a number of imaged molecules unequivocally by simultaneously recording their fluorescence emission spectra. Furthermore, their spectral characteristics not only allow us to separate different fluorescent labels quantitatively and qualitatively but also provide information on the microenvironment of the molecules. This new method was successfully tested on a system of yellow-green and red fluorescent 20-nm latex beads, and its usefulness in studies of biological systems was illustrated for a preparation of combined binary ratio labeling-fluorescence in situ hybridization-stained mouse chromosomes.