Unusual features of the c-ring of F1FO ATP synthases.

Membrane integral ATP synthases produce adenosine triphosphate, the universal "energy currency" of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it.

Creation and study of triterpenoid nanoparticles and radioprotective substance genistein.

This work is devoted to the study and obtaining of new radioprotective agents based on natural flavonoid genistein and spherical amorphous nanoparticles (SANPs) produced from a mixture of birch bark triterpenoids. The physicochemical characteristics of the nanoparticles were studied by electron microscopy, dynamic light scattering, and UV-VIS spectroscopy. The radioprotective efficacy of the nanodrug in vivo and the possibility of its use as a radioprotective agent was shown.

[Creation and Study of Triterpenoid Nanoparticles and Amphiphilic meso-Arylporphyrins].

This work is devoted to the study of nanoparticles based on amphiphilic meso-arylporphyrins and spherical amorphous nanoparticles (SANp), consisting of birch bark triterpenoids mixture. Nanoparticles were investigated by electron microscopy, dynamic light scattering, UV spectroscopy and fluorimetry. It was shown the efficiency of the inclusion of porphyrin sensitizer to the nanoparticles and the use of these nanoparticles as drug delivery system.

The role of chain rigidity in lipid self-association: comparative study of dihexanoyl- and disorbyl-phosphatidylcholines.

In the course of structure-function investigations of lipids a phosphatidylcholine molecule with short and rigid tails, di-2,4-hexadienoylphosphatidylcholine (DiSorbPC), was synthesized and studied in comparison with its saturated analog, dihexanoylphosphatidylcholine (DHPC). Conjugated double bonds in the acyl chains in DiSorbPC reduce considerably the number of possible conformers of the lipid within an aggregate. This leads to impaired packing of unsaturated acyl chains and thus, to a surprisingly high (115 Å(2)) area per molecule for DiSorbPC at the air-water interface and failure to form micelles of regular size and shape. Details on DiSorbPC aggregation and packing provided by a set of experimental techniques combined with molecular dynamics simulations are presented.

Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments.

Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.

Bacterial Synthesis and Purification of Normal and Mutant Forms of Human FGFR3 Transmembrane Segment.

The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their(15)N- and [(15)N,(13)C]-isotope-labelled derivatives, were described. Each peptide was produced inEscherichia coliBL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.

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.

Expression of G-protein coupled receptors in Escherichia coli for structural studies.

To elaborate a high-performance system for expression of genes of G-protein coupled receptors (GPCR), methods of direct and hybrid expression of 17 GPCR genes in Escherichia coli and selection of strains and bacteria cultivation conditions were investigated. It was established that expression of most of the target GPCR fused with the N-terminal fragment of OmpF or Mistic using media for autoinduction provides high output (up to 50 mg/liter).

Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels.

An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives ((15)N-, (15)N-/13C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly alpha-helical conformation. The existence of cross-peaks for all glycines of the (15)N-HSQC NMR spectra as well as relatively small line widths (~20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.

Lipid-protein nanodiscs: possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides.

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.

Mega-ampere submicrosecond generator GIT-32.

The GIT-32 current generator was developed for experiments with current carrying pulsed plasma. The main parts of the generator are capacitor bank, multichannel multigap spark switches, low inductive current driving lines, and central load part. The generator consists of four identical sections, connected in parallel to one load. The capacitor bank is assembled from 32 IEK-100-0.17 (0.17 microF, 40 nH, 100 kV) capacitors, connected in parallel. It stores approximately 18 kJ at 80 kV charging voltage. Each two capacitors are commuted to a load by a multigap spark switch with eight parallel channels. Switches operate in ambient air at atmospheric pressure. The GIT-32 generator was tested with 10, 15, and 20 nH inductive loads. At 10 nH load and 80 kV of charging voltage it provides 1 MA of current amplitude and 490 ns rise time with 0.8 Omega damping resistors in discharge circuit of each capacitor and 1.34 MA530 ns without resistors. The net generator inductance without a load was optimized to be as low as 12 nH, which results in extremely low self-impedance of the generator ( approximately 0.05 Omega). It ensures effective energy coupling with low impedance loads like Z pinch. The generator operates reliably without any adjustments in 40-80 kV range of charging voltage. Maximum jitter (relative to a triggering pulse) at 40 kV charging voltage is about 7 ns and lower at higher charging voltages. Operation and handling are very simple, because no oil and no purified gases are required for the generator. The GIT-32 generator has dimensions of 3200 x 3200 x 400 mm(3) and total weight of about 2500 kg, thus manifesting itself as a simple, robust, and cost effective apparatus.

2H-NMR comparative study of phosphocholine group conformation in bilayers composed of diacyl and dialkyl phosphatidylcholines.

Model membranes formed from 1,2-dihexadecyl-, 1,2-dipalmitoyl-, 1,2-ditetradecyl- or 1,2-dimyristoyl-rac-glycero-3-phosphocholine, deuterium-labelled at choline groups, in an excess of water were compared using 2H-NMR spectroscopy. The dynamics and conformation of the labelled choline segments were estimated based on spin-lattice relaxation time and residual quadrupole splittings. The trimethylammonium group of dialkyl phosphatidylcholine was shown to be more distant from the bilayer surface as compared with that of the diacyl analogues.

[Lipid inhibitors of phospholipase A2]. / Lipidnye ingibitory fosfolipazy A2.

Results of studies of lipid inhibitors of phospholipase A2 are reviewed with a special emphasis on substrate specificity, special features of interfacial catalysis, and methods for the determination of the enzyme activity. The biological function of intracellular phospholipases is considered, and the possible use of inhibitors of a lipid nature for the modulation of the enzyme activity in pathological states of the human organism is discussed.

On the synthesis of platelet-activating factor via acetylation of 1-alkyl-sn-glycero-3-phosphocholine. Formation of structural isomer of PAF in the presence of bases.

It has been shown that platelet-activating factor (PAF) specimens prepared via acetylation of 1-alkyl-sn-glycero-3-phosphocholine (lyso-PF) with acetic anhydride are heterogeneous. The contaminated compound was isolated and identified to be the structural isomer of PAF, 1-alkyl-3-acetyl-sn-glycero-2-phosphocholine (iso-PAF). It appeared, that iso-PAF is formed when performing the reaction in the presence of organic bases,but not under acid catalysis. The mechanism of iso-PAF formation is discussed.

Regulation of lipid polymorphism is essential for the viability of phosphatidylethanolamine-deficient Escherichia coli cells.

Escherichia coli strain AD93 is unable to synthesize the nonbilayer lipid phosphatidylethanolamine and requires high concentrations of specific divalent cations for growth. Previous studies suggested that in this strain, cardiolipin in combination with divalent cations functionally replaces phosphatidylethanolamine, reflecting polymorphic regulation of membrane lipid composition. However, it is also possible that divalent cations are required for regulation of lipid packing or membrane surface potential. 2H NMR was employed to measure the effect of different divalent cations on lipid packing in aqueous dispersions of lipid extracts isolated from AD93 and the wild type parental strain W3899, which were grown with [11,11-2H2]oleic acid. The results indicate that a range of acyl chain order is compatible with growth and that Ba2+, which cannot support growth of AD93, can increase chain packing to the wild type level. By means of microelectrophoresis, it was shown that the growth-promoting cations and Ba2+ have a strong and comparable ability to screen the surface charge of large unilamellar vesicles prepared from AD93 lipid extracts. Therefore, it is unlikely that the growth-promoting capacity of divalent cations is primarily due to their effect on lipid packing or their potency to decrease the surface potential. Furthermore, the addition of small amounts of Ba2+ to a AD93 lipid dispersion with excess Mg2+ diminished HII phase formation. This observation can explain the growth arrest in AD93 cultures upon the addition of Ba2+ and further supports the conclusion that the cation requirement of this strain arises mainly from polymorphic regulation of lipid composition.

[Formation of a structural isomer of platelet activating factor during acetylation of 1-alkyl-sn-glycero-3-phosphocholine]. / Obrazovanie strukturnogo izomera faktora aktivatsii trombotsitov pri atsetilirovanii 1-alkil-sn-glitsero-3-fosfokholina.

In studying acetylation of 1-alkyl-sn-glycero-3-phosphocholine (lyso PAF) with acetic anhydride, a key step of the platelet activating factor (PAF) synthesis, we have found that in the presence of triethylamine or 4-dimethylaminopyridine some 1-alkyl-3-acetyl-sn-glycero-2-phosphocholine as an admixture to PAF formed, whereas the acid catalysed reaction resulted in isomerically pure PAF. The mechanism of the reaction leading to the PAF structural isomer is discussed.

[Study of the interaction of alpha-tocopherol with phospholipids, fatty acids, and their oxygenated derivatives by (31)P-NMR spectroscopy]. / Izuchenie vzaimodeistviia alpha-tokoferola s fosfolipidami, zhirnymi kislotami i ikh oksigenirovannymi proizvodnymi metodom (31)P-IaMR- spektroskopii.

Interaction of alpha-tocopherol with phospholipids, oleic, ricinoleic acids and linoleic acid hydroperoxides was investigated by means of 31P NMR spectroscopy on a model artificial membranes containing egg phosphatidylcholine and lysophosphatidylcholine. alpha-Tocopherol was shown to support the bilayer organization of lysophospholipids, whereas its introduction into the lecithin-water system stimulated the hexagonal phase formation. Free fatty acids exhibited a synergism to alpha-tocopherol, the effect of the hexagonal phase formation being at most increased by oxygenated acids--ricinoleic acid and linoleic acid hydroperoxides. In accordance with the experimental data, a conclusion about modifying and structuring action of alpha-tocopherol was made. Origin of the alpha-tocopherol's modulating effect on the membrane structure and a possible role of hexagonal phase forming upon its action in the course of peroxidation of lipids was discussed.

[Immunomodulating properties of phospholipids]. / Izuchenie immunomoduliruiushchikh svoistv fosfolipidov.

Immunomodulating properties of glycerophospholipids were studied. Both phosphatidyl ethanolamines and phosphatidyl cholines exhibited similar immunostimulating effects.