3 research outputs found
Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins
Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the singleliposome level and for the first time apply it to study photosynthetic pigment protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment
Light Harvesting in a Fluctuating Antenna
One of the major players in oxygenic
photosynthesis, photosystem
II (PSII), exhibits complex multiexponential fluorescence decay kinetics
that for decades has been ascribed to reversible charge separation
taking place in the reaction center (RC). However, in this description
the protein dynamics is not taken into consideration. The intrinsic
dynamic disorder of the light-harvesting proteins along with their
fluctuating dislocations within the antenna inevitably result in varying
connectivity between pigment–protein complexes and therefore
can also lead to nonexponential excitation decay kinetics. On the
basis of this presumption, we propose a simple conceptual model describing
excitation diffusion in a continuous medium and accounting for possible
variations of the excitation transfer rates. Recently observed fluorescence
kinetics of PSII of different sizes are perfectly reproduced with
only two adjustable parameters instead of the many decay times and
amplitudes required in standard analysis procedures; no charge recombination
in the RC is required. The model is also able to provide valuable
information about the structural and functional organization of the
photosynthetic antenna and in a straightforward way solves various
contradictions currently existing in the literature
Exciton Band Structure in Bacterial Peripheral Light-Harvesting Complexes
The variability of the exciton spectra of bacteriochlorophyll
molecules
in light-harvesting (LH) complexes of photosynthetic bacteria ensures
the excitation energy funneling trend toward the reaction center.
The decisive shift of the energies is achieved due to exciton spectra
formation caused by the resonance interaction between the pigments.
The possibility to resolve the upper Davydov sub-band corresponding
to the B850 ring and, thus, to estimate the exciton bandwidth by analyzing
the temperature dependence of the steady-state absorption spectra
of the LH2 complexes is demonstrated. For this purpose a self-modeling
curve resolution approach was applied for analysis of the temperature
dependence of the absorption spectra of LH2 complexes from the photosynthetic
bacteria <i>Rhodobacter (Rba.) sphaeroides</i> and <i>Rhodoblastus (Rbl.) acidophilus</i>. Estimations of the intradimer
resonance interaction values as follows directly from obtained estimations
of the exciton bandwidths at room temperature give 385 and 397 cm<sup>–1</sup> for the LH2 complexes from the photosynthetic bacteria <i>Rba. sphaeroides</i> and <i>Rhl. acidophilus</i>,
respectively. At 4 K the corresponding couplings are slightly higher
(391 and 435 cm<sup>–1</sup>, respectively). The retained exciton
bandwidth at physiological conditions supports the decisive role of
the exciton coherence determining light absorption in bacterial light-harvesting
antenna complexes