Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum.

One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.

Predicted bacteriorhodopsin from Exiguobacterium sibiricum is a functional proton pump.

The predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.

[Ion channels of various types induced in lipid membranes by gramicidin A derivatives carrying a cationic sequence at their C-termini].

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both beta6.3 single-stranded and beta5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.

[Synthesis and properties of the retro-analogue of myelopeptide MP-2].

The bone marrow myelopeptide MP-2 (Leu-Val-Val-Tyr-Pro-Trp), exhibiting antitumor activity, and its retro-analogue (Trp-Pro-Tyr-Val-Val-Leu) were synthesized, and their properties were studied. The in vitro and in vivo activities of retro-MP-2 were comparable with those of MP-2. Both peptides equally restored the functional activity of T-lymphocytes inhibited by toxins released by HL-60 cells and inhibited by 70-82% the growth of various types of transplantable solid tumors: Ca-755 adenocarcinoma of the mammary gland, Lewis adenocarcinoma of the lung, and S180 sarcoma. The positions and intensities of the Cotton effects in CD spectra of the MP-2 peptide and its retro-analogue in various solvents are almost indistinguishable. The positions of extrema and integral intensities of the amide I and amide A bands in IR spectra of both peptides were practically identical. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

Structural polymorphism of gramicidin A channels: ion conductivity and spectral studies.

The relation between the various spatial structures of the gramicidin A channels and their ionic conductance has been studied. For this aim, various conformations of the peptide were pre-formed in liposomal bilayer and after subsequent fusion of liposomes with planar lipid bilayer the measured channel conductance was correlated with gramicidin structures established in liposomes. To form the single-stranded pi 6.3 pi 6.3 helix the peptide and lipid were co-dissolved in TFE prior to liposome preparation. THF and other solvents were used to form parallel and antiparallel double helices. Conformation of gramicidin in liposomes made by various phosphatidylcholines was monitored by CD spectroscopy, and computer analysis of the spectra obtained was performed. After fusion of gramicidin containing liposomes with planar bilayer membranes from asolectin, the histograms of single-channel conductance were obtained. The histograms had one or three distinct peaks depending on the liposome preparation. Assignment of the structure of the channel to conductance levels was made by correlation of CD data with conductance histograms. The channel-forming analogue, des(Trp-Leu)2-gramicidin A, has been studied by the same protocol. The channel conductances of gramicidin A and the shortened analogue increase in the following order: antiparallel < parallel < pi 6.3 pi 6.3. Single-channels formed by double helices have higher dispersity of conductance than the pi 6.3 pi 6.3 helical channel. Lifetimes of the double helical and the pi 6.3 pi 6.3 helical channels are very close to each other. The data obtained were compared with theoretically predicted properties of double helices [1].

The double pi pi 5.6 helix of gramicidin A predominates in unsaturated lipid membranes.

The structure of the channel-forming polypeptide gramicidin A (GA) incorporated into phosphatidyl-choline (PC) liposomes has been studied as a function of the degree of unsaturation of the acyl chains of PC. The initial conformational state of GA in reconstituted bilayers is determined by the solvent in which the peptide and the lipid are initially co-dissolved, whereas the equilibrium conformational state (after heat incubation) is affected by the lipid structure rather than by the nature of the solvent. The conformational equilibrium of GA has been studied in liposomes prepared from PC having a variable number of double bonds in the fatty acid moiety, by circular dichroism and Fourier transform infrared. Liposomes were prepared from trifluoroethanol or ethanol solutions and incubated at 68 degrees C. GA was shown to retain the conformation of the right-handed pi-->6.3 pi<--6.3 helix in PC with saturated acyl chains and with one double bond, whereas in dilinoleoyl-PC, having two double bond in each chain, the thermodynamically preferred structures are left-handed antiparallel and parallel double pi pi 5.6 helices. Natural soybean PC also favours left-handed pi pi 5.6 helical structures of GA (approximately 75%). This finding is discussed in terms of the role of PC unsaturation in the dynamic properties of the lipid matrix. Differences between observed FTIR spectra of the increases decreases pi pi 5.6 helix in solution (and to a larger extent in the membrane) and the calculated IR spectra can be interpreted as resulting from deviation of the real structure from the theoretically derived ideal helix. The data obtained provide grounds for better understanding of a GA channel functioning in lipids of variable degrees of unsaturation.

[Epidemiological effectiveness of an inactivated three-component flu vaccine with an increased concentration of hemagglutinin in the inoculation dose]. / Epidemiologicheskia éffektivnost' inaktivirovannoi trekhkomponentnoigrippoznoi vaktsiny s povyshennoi kontsentratsiei gemaggliutinina v privivochnoi doze.

The work presents the results of the evaluation of mass immunization of working adults with inactivated trivalent influenza vaccine under the conditions of an epidemic caused by influenza viruses A (H1N1), A (H3N2) and B. This immunization produced no effect on influenza morbidity in the groups of vaccinees in comparison with those of nonvaccinated persons. The index of effectiveness was 1.0 and less. The ineffectiveness of mass immunization was due to a high level of natural immunity to influenza and the extensive use of influenza vaccine in past years.

Effectiveness of peroral and intranasal immunization of school children with live allantoic influenza vaccine- and comparative study.

A comparative study of two preparations of allantoic live influenza vaccine, one for intranasal and the other for peroral immunization of children of school age, was peformed under conditions of a blind epidemiological trial. Previously obtained data on the safety and high immunogenicity of the intranasal vaccine variant, prepared from extremely attenuated cold-adapted strains, were confirmed. The peroral administration of the live influenza vaccine, in use in the USSR for active immunization of the adult population, also stimulated influenza immunity without producing postvaccinal reactions. Peroral and intranasal immunization with the above variants of live allantoic influenza vaccine markedly lowered in the frequency of influenza disease during an influenza epidemic, the mean index of effectiveness being equal to a factor of 2. Evidence of prospectiveness of influenza prophylaxis among school children was obtained.