Hydrophobicity and prediction of the secondary structure of membrane proteins and peptides.

Reliability of the hydropathy method to predict the formation of membrane-spanning alpha-helices by integral membrane proteins and peptides whose structure is known from X-ray crystallography is analysed. It is shown that Kyte-Doolittle hydropathy plots do not predict accurately 22 transmembrane alpha-helices in the reaction centres (RC) of the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides (R-26). The accuracy of prediction for these proteins was improved using an optimised Kyte-Doolittle hydrophobicity scale. However, this hydrophobicity scale did not improve the predictions for the alphabeta-peptides of the B800-850 (LH2) complexes of the photosynthetic bacteria Rhodopseudomonas acidophila and Rhodospirillum molischianum, which were excluded from the optimisation procedure. The best and worst predictions of membrane-spanning alpha-helices for the RC proteins and LH2 peptides, respectively, were obtained with a propensity scale (PRC) calculated from the amino acid sequences and X-ray data for the RC proteins. A propensity scale (PLH) obtained using the amino acid sequences and X-ray data for the alphabeta-peptides of the LH2 complexes did not give an acceptable prediction of the transmembrane segments in the LH2 peptides; moreover, it markedly contradicted the PRC scale. Amino acids have been concluded to have no significant preference to localisation in transmembrane segments. Therefore, the predictive ability of the hydropathy methodology appears to be limited: the number of transmembrane segments can be correctly calculated for the best case only, and the lengths and positions of membrane-spanning alpha-helices in a protein amino acid sequence can not be predicted exactly.

On the influence of pigment-protein interactions on energy transfer processes in photosynthetic membrane structures. 3. The FMO complex of Chlorobium tepidum at high pressure.

The low-temperature absorption spectra of the Chlorobium tepidum FMO bacteriochlorophyll-protein complex at various pressures have been calculated within the framework of mini-exciton theory. The dependences of the Qy transition energies of the monomeric pigments on pressure have been found by means of functional minimization. This functional includes the parameters of both theoretical and experimental absorption spectra at low temperatures and various pressures. The dependences obtained are compared with those derived for the exciton transition energies, which have been obtained by deconvoluting absorption spectra with seven Gaussian components at each pressure. The pressure increase has been shown to result in the increased coupling energy between both the pigment molecules themselves and pigments and amino acid residues. The pigment molecules capable of binding histidines and water molecules have been shown to have the greatest and smallest responses to increased pressure, respectively. The couplings of Bchl molecules with the surrounding amino acid residues have been shown to change both the exciton delocalization index and the exciton distribution between the pigment molecules within the protein subunit; the increased pressure does not change these parameters significantly.

On the influence of pigment-protein interactions on the energy transfer processes in photosynthetic membrane structures. 2. LH2 complex of Rhodobacter sphaeroides.

Low-temperature heterogeneous absorption and circular dichroism spectra of the Rb. sphaeroides LH2 complexes are calculated within the framework of the mini-exciton theory and diagonal static random disorder for the pure electronic transitions of the monomeric Bchl molecules. The coupling of Bchl molecules with the surrounding amino acid residues has been shown to change both the exciton distribution between the pigment molecules in each of the exciton states. The value of the delocalization index depends on the excitation wavelength and varies between 2-6 Bchl molecules. The optical transitions occurring at 780-790 and 820 nm have been found to be strongly mixed so that all Bchl molecules of the LH2 complex predetermine absorption in these spectral regions. On the other hand, absorption at 800 and 850 nm is mainly determined by the cycles of 9 and 18 Bchl molecules, respectively. Thus, the light energy absorbed by the B800 molecules at 800 nm is transferred to the B850 molecules by the interlevel exciton relaxation processes due to the population of the heavily mixed 820-nm exciton levels. The width of the heterogeneous absorption band for the cyclic monomeric aggregate has been shown to decrease as compared with the monomeric absorption band by square root(Ndel) time, where Ndel is the mean number of pigments over which the exciton is delocalized within the excited absorption band.

The structural organization of the antenna chromophore protein complexes in membranes of the photosynthetic bacterium Rhodopseudomonas viridis.

A possible structural and functional organization of the antenna chromophore protein complexes (CPC) in the Rhodopseudomonas viridis membranes was considered in terms of structural models proposed by Zuber and Brunisholz (in Chlorophylls, ed. H. Scheer (Boca Raton: CRC Press, 1991):626-703). Analysis of the absorption spectra led to the conclusion that the number of the antenna bacteriochlorophyll molecules per reaction center (RC) is 30 +/- 3 both for chromophores and quantasomes of Rps. viridis. It implies a multicentral organization of the CPCs around RCs, when the CPC of cyclic structure is formed by (alpha beta gamma)4 polypeptides. A multicentral model predicts an almost linear dependence of the antenna fluorescence yield on the oxidized primary donor concentration if the antenna fluorescence lifetimes are assumed to be 60-70 and 110-120 ps for the open and closed RCs, respectively, which is in agreement with the experimental observations. We conclude that the Rps. viridis membrane domain consists of 4-6 RCs surrounded by 6-22 CPCs, and both of these protein subsystems are packed into a hexagonal array.

On the influence of pigment-protein interactions on the energy transfer processes in photosynthetic membrane structures.

Low-temperature heterogeneous absorption and circular dichroism spectra of the Prosthecochloris aestuarii FMO complex were calculated within the framework of the mini-exciton theory including both the inhomogeneous distribution of exciton line frequencies and static random disorder of the pure electronic transitions of Bchl molecules. The frequencies of the Qy pure electronic transitions of Bchl molecules immobilized by the FMO complex polypeptides were found by minimization of a functional which links the parameters of the theoretical and experimental optical spectra. The interactions of Bchl molecules with surrounding amino acid residues was shown to change both the exciton delocalization index and exciton distribution between the pigment molecules in each exciton energetic state. As a consequence, the interlevel exciton relaxation processes, being accompanied by essential changes in the exciton distribution between pigment molecules, lead to the energy transfer within the FMO complex. The model spectra calculations within the framework of the static random disorder approach were shown to give unacceptable results.

Thermoluminescence in photosynthesis. 4. Influence of energy transfer processes.

Thermoluminescence (TL) emission arising from charge recombination in photosynthetic reaction centres was examined considering the energy transfer between antenna chlorophylls and the reaction centre. The energy transfer processes were shown to decrease the width and change the temperature of the maximum glow of the TL band profiles when the rate constant of direct recombination between the oxidized primary electron donor and reduced primary acceptor (pheophytin or bacteriopheophytin) did not exceed 10(6) s-1. The primary radical pair lifetime of 3.10(-9)-10(-8) s was shown to minimize the influence of energy transfer on the TL band profiles and, therefore, the energy transfer can be neglected when the TL band profiles are calculated. The profile of the TL band arising from charge recombination in the photosystem 2 state S2QB.- was shown to depend considerably on the temperature-dependent rate constant of direct recombination between QB.- and S2 state of the photosystem 2 donor site.

[Estimation of the number of manganese atoms functioning in the donating side of photosystem 2]. / Opredelenie chisla atomov margantsa, funktsioniruiushchikh v donornoi chasti fotosistemy II.

Comparison of theoretically expected and experimental dependence of reactivation of the donor side of photosystem 2 (PS-2) on concentration of exogenic MnCl2 that was observed in PS-2 preparation after complete extraction of manganese was made. It is shown that in the absence of Mg2+ or bivalent cations of other metals (Me2+) the reactivation corresponds to the filling of a tetranuclear Mn-containing active center; in the presence of 3 microM Mg2+ or other Me2+ the reactivation takes place as a result of binding of two manganese atoms in the center. It is suggested that a tetranuclear manganese complex (which probably is responsible for the photosynthetic oxidation of water) functions in the donor side of PS-2; two of these four manganese atoms can be substituted by Mg2+ or other Me2+ without changing activity of the center.

[Quantum yield formation of triplet state and recombination luminescence of the primary electron donor in reaction centers of photosynthetic bacteria]. / O kvantovom vykhode tripletnykh sostoianii i rekombinatsionnoi liuminestsentsii pervichnogo donra élektrona v reaktsionnykh tsentrakh fotosinteziruiushchikh bakterii.

The quantum yield of fluorescence and triplet state of the primary electron donor (P) in reaction centers of the photosynthetic bacteria under reduction conditions (quinon - Fe complex is reduced) are discussed. The kinetics of the reversible electron transfer in ion radical pair [P+ primary acceptor -.] are considered by means of the ordinary kinetic equations and of the nonequilibrium spin-density operator method. It is shown, that present experimental data is not sufficient to find the value of energy (delta E) dissipated in the process of primary charge separation. The range of admissible values delta E can be defined on the base of the temperature dependence of the P fluorescence under reduction conditions.

[Optimization of isolation conditions for three types of pigment protein-lipid complexes from chloroplasts during solubilization with Triton X-100]. / Optimizatsiia uslovii vydeleniia trekh tipov pigment-belkovolipidnykh kompleksov khloroplastov gorokha pri soliubilizatsii s pomoshch'iu tritona X-100.

The maximal total release of pigment protein-lipid complexes (PPLC) during their isolation from pea chloroplasts was achieved by 1-hr solubilization with Triton X-100, the Triton:chlorophyll (T/Chl) ratio being 50 mg/mg/ml. The total yield of the reaction center complexes (sigma PPLC RC) was 22,3%, whereas that of the auxiliary light-accumulating complex (ALA-PPLC) was approximately 32% with respect to Chl. An increase in the solubilization time and of the T/Chl ratio resulted in dissociation of ALA-PPLC. On the contrary, the reaction center complexes steadily maintained their composition and high photochemical activity within a wide range of T/Chl during 24--28 hrs of solubilization. The purest preparations of PPLC RC of phostosystem I (PS-I) were obtained by 24 hr-incubation (T/Chl = 80); their Chl/P700 ratio after a single fractionation on DEAE-cellulose was equal to 36. A considerable increase of T/Chl and of the solubilization time hampered the chromatographical separation of PPLC RC of PS-I and PPLC RC of PS-II. The optimal conditions for isolation of PPLC RC of PS-I and PPLC RC of PS-II were: solubilization at T/Chl 80--120 and prolongation of incubation time from 5 to 7 hrs. The photochemical activity of the complexes obtained was maximal and correlated with the minimal content of admixture P700 (1 molecule of P700 per 450--500 molecules of Chl.).

[Photoreduction of bacteriophenophytin b in the primary light reaction of Rhodopseudomonas viridis chromatophores]. / Fotovosstanovlenie bakteriofeofitina b v pervichnoi svetovoi reaktsii khromatoforov Rhodopseudomonas viridis

Photoconversions of the reaction center pigments in chromatophores of nonsulfur purple bacteria Rhodopseudomonas viridis have been studied as a function of redox potential of medium (Eh). It has been shown that at a decrease in the Eh values from +400 mV to--100 divided by--600 mV a photo-induced accumulation of P980+ (oxidized primary electron donor in R. viridis) is replaced by the photoaccumulation of a reduced pigment complex P800 (bleaching of bacteriopheophytin b absorption bands at 545 and 800 nm, a development of broad bands at 680 and 430 nm and a blue shift of the bacteriochlorophyll band at 830 nm). The P800 photoreduction is observed under illumination by light with lambda greater than 900 nm between +20 divided by--196 degrees C at pH 3,5--12,5 and is accompanied by oxidation of the cytochrome and an increase in fluorescence yield of bacteriochlorophyll. It is suggested that P800 accepts an electron from the P980 in the primary photoreaction, which preceeds ubiquinone reduction. A midpoint redox potential (Em) is found to be of --620(+/- 20) mV for the P800/P800- and +515 (+/-20) mV for the P980/+P980. At a decrease in the Eh value down to -400 mV luminescence has been detected with T1/2 8 nsec, an activation energy of 0,065 +/- 0,02 ev and quantum yield being close to the fluorescence yield. It is assumed that this luminescence is a result of charge recombination in the biradical P980+ -- P800-.