Effect of Dipyridamole on Membrane Energization and Energy Transfer in Chromatophores of Rba. sphaeroides.

Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of photosynthetic electron transport chain in the bacterial chromatophores of Rba. sphaeroides was investigated. DIP was found to affect efficiency of energy transfer from the light-harvesting complex LH2 to the LH1-reaction center core complex and to produce the long-wavelength ("red") shift of the absorption band of light-harvesting bacteriochlorophyll molecules in the IR spectral region at 840-900 nm. This shift is associated with the membrane transition to the energized state. It was shown that DIP is able to reduce the photooxidized bacteriochlorophyll of the reaction center, which accelerated electron flow along the electron transport chain, thereby stimulating generation of the transmembrane potential on the chromatophore membrane. The results are important for clarifying possible mechanisms of DIP influence on the activity of membrane-bound functional proteins. In particular, they might be significant for interpreting numerous therapeutic effects of DIP.

Comparison of tryptophan fluorescence lifetimes in cyanobacterial photosystem I frozen in the light and in the dark.

The dependence on temperature of tryptophan fluorescence lifetime in trimeric photosystem I (PSI) complexes from cyanobacteria Synechocystis sp. PCC 6803 during the heating of pre-frozen to - 180 °C in the dark or in the light-activated preparations has been studied. Fluorescence lifetime in samples frozen in the light was longer than in samples frozen in the dark. For samples in 65% glycerol at λreg = 335 nm and at 20 °C, the lifetime of components were as follows: τ1 ≈ 1.2 ns, τ2 ≈ 4.9 ns, and τ3 ≈ 20 ns. The contribution of the first component was negligible. To analyze the contribution of components 2 and 3 derived from frozen-thawed samples, two temperature ranges from - 180 to - 90 °C and above - 90 °C are considered. In doing so, the contributions of these components appear antiphase course to each other. The dependence on temperature of these contributions is explained by the influence of the microconformational protein dynamics on the tryptophan fluorescence lifetime. In the present work, a comparative analysis of temperature-dependent conformational dynamics and electron transfer in cyanobacterial PSI (Schlodder et al., in Biochemistry 37:9466-9476, 1998) and Rhodobacter sphaeroides reaction center complexes (Knox et al., in J Photochem Photobiol B 180:140-148, 2018) was also carried out.

The effect of light and temperature on the dynamic state of Rhodobacter sphaeroides reaction centers proteins determined from changes in tryptophan fluorescence lifetime and P+QA- recombination kinetics.

The temperature dependencies of the rate of dark recombination of separated charges between the photoactive bacteriochlorophyll and the primary quinone acceptor (QA) in photosynthetic reaction centers (RCs) of the purple bacteria Rhodobacter sphaeroides (Rb. sphaeroides) were investigated. Measurements were performed in water-glycerol and trehalose environments after freezing to -180 °C in the dark and under actinic light with subsequent heating. Simultaneously, the RC tryptophanyl fluorescence lifetime in the spectral range between 323 and 348 nm was measured under these conditions. A correlation was found between the temperature dependencies of the functional and dynamic parameters of RCs in different solvent mixtures. For the first time, differences in the average fluorescence lifetime of tryptophanyl residues were measured between RCs frozen in the dark and in the actinic light. The obtained results can be explained by the RC transitions between different conformational states and the dynamic processes in the structure of the hydrogen bonds of RCs. We assumed that RCs exist in two main microconformations - "fast" and "slow", which are characterized by different rates of P+ and QA- recombination reactions. The "fast" conformation is induced in frozen RCs in the dark, while the "slow" conformation of RC occurs when the RC preparation is frozen under actinic light. An explanation of the temperature dependencies of tryptophan fluorescence lifetimes in RC proteins was made under the assumption that temperature changes affect mainly the electron transfer from the indole ring of the tryptophan molecule to the nearest amide or carboxyl groups.

NMR spin-echo studies of hydration properties of the molecular chaperone alpha-crystallin in the bovine lens.

The water-binding properties of bovine lens alpha-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin-echo technique was used to study the hydration of protein samples and to determine the spin-spin relaxation times (T2) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P(0)=0.75, alpha-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P(0)=1.0, the absorption of water by alpha-crystallin was 17% with a single component decay character of spin-echo (T2=3 ms). Addition of water to alpha-crystallin to about 50% of its w/w in the protein sample showed T2=8 ms with only one single component decay of the spin-echo signal. The single component decay character of the spin-echo indicates at the tightly bound water by alpha-crystallin. Under a relative humidity P/P(0)=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T2 of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with alpha-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for alpha-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that alpha-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of alpha-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.