Fluorescence Spectroscopy of Conformational Changes of Single LH2 Complexes

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm. The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The probability for a particle to undergo significant spectral shift in either direction was found to be roughly the same. Using the modified Redfield theory, the observed changes in spectral shape and intensity were accounted for by changes in the realization of the static disorder. Long lifetimes of the quasi-stable states suggest large energetic barriers between the states characterized by different emission spectra

Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms

Characterization of thymine microcrystals by CARS and SHG microscopy

Identification of chemically homologous microcrystals in a polycrystal sample is a big challenge and requires developing specific highly sensitive tools. Second harmonic (SHG) and coherent anti-Stokes Raman scattering (CARS) spectroscopy can be used to reveal arrangement of thymine molecules, one of the DNA bases, in microcrystalline sample. Strong dependence of CARS and SHG intensity on the orientation of the linear polarization of the excitation light allows to obtain high resolution images of thymine microcrystals by additionally utilizing the scanning microscopy technique. Experimental findings and theoretical interpretation of the results are compared. Presented experimental data together with quantum chemistry-based theoretical interpretation allowed us to determine the most probable organization of the thymine molecules

Spectral Dynamics of Individual Bacterial Light-Harvesting Complexes: Alternative Disorder Model

The bacterial (Rhodopseudomonas acidophila) photosynthetic peripheral light-harvesting complex of type 2 (LH2) exhibits rich fluorescence spectral dynamics at room temperature. The fluorescence spectrum of individual LH2 shifts either to the blue or to the red during the experimental observation time of a few minutes. These spectral changes are often reversible and occur between levels of a distinctly different peak wavelength. Furthermore, they are accompanied by a change of the spectral line shape. To interpret the dynamics of spectral changes, an energetic disorder model associated with easily explainable structural changes of the protein is proposed. This model assumes that each pigment in the tightly coupled ring of bacteriochlorophylls can be in two states of electronic transition energy due to the protein-pigment interaction. The transition between these structural, and hence spectroscopic, states occurs through the thermally induced conformational potential energy barrier crossing. Although simplified, the model allows us to reproduce the bulk fluorescence spectrum, the distribution of the single-molecule spectral peak wavelength and its changes, and the statistics of the duration of the spectral states. It also provides an intuitively clear picture of possible protein dynamics in LH2. At the same time, it requires additional sophistication since it essentially does not reproduce the red occurrences of single LH2 spectra