Alexander L. Ksenofontov

Interaction of tumor and normal blood cells with ethylene oxide and propylene oxide block copolymers

Ethylene oxide and propylene oxide block copolymers (pluronics) are widely known as agents that promote drug penetration across biological barriers. We have studied the interaction of normal and malignant blood cells with pluronics L61 and P85 that have different hydrophobicity. SP2/0 myeloma cells accumulated pluronics while normal cells adsorb most of the polymer on the surface. Interaction of pluronics with cells resulted in drastic changes of membrane microviscosity. Tumor cell membrane microviscosity decreased after pluronics adsorption, in contrast to normal cells, whose membrane microviscosity was enhanced. We suppose that sensitivity of tumor cell membrane microviscosity to the pluronics action correlates with its permeability for molecular substances.

Structural Analysis of Influenza A Virus Matrix Protein M1 and Its Self-Assemblies at Low pH

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

Increase in the specific radioactivity of tritium-labeled compounds obtained by tritium thermal activation method

Abstract A method of tritium introduction into different types of organic molecules that is based on the interaction of atomic tritium with solid organic target is described. Tritium atoms are formed on the hot W-wire, which is heated by the electric current. Such an approach is called “tritium thermal activation method”. Here we summarize the results of labeling globular proteins (lysozyme, human and bovine serum albumins); derivatives of pantothenic acid and amino acids; ionic surfactants (sodium dodecylsulfate and alkyltrimethylammonium bromides) and nonionic high-molecular weight surfactants – pluronics. For the first time it is observed that if the target-compound is fixed and its radicals are stable the specific radioactivity of the labeled product can be drastically increased (up to 400 times) when the target temperature is ca. 295 K compared with the results obtained at 77 K. The influence of labeling parameters as tritium gas pressure, exposure time and W-wire temperature was tested for each target temperature that results in the optimum labeling conditions with high specific radioactivity and chemical yield of the resulting compound.

Tritium planigraphy study of structural alterations in the coat protein of Potato virus X induced by binding of its triple gene block 1 protein to virions

Alterations in Potatou2003virusu2003X (PVX) coat protein structure after binding of the protein, encoded by the first gene of PVX triple gene block (triple gene blocku20031 protein, TGBp1), to the virions were studied using tritium planigraphy. Previously, it has been shown that TGBp1 molecules interact with the PVX particle end, containing the 5′‐terminus of PVX RNA, and that this interaction results in a strong decrease in virion stability and its transformation to a translationally active state. In this work, it has been shown that the interaction of TGBp1 with PVX virions leads to an increase of ∼u200350% in tritium label incorporation into the 176–198 segment of the 236‐residue‐long PVX coat protein subunit, with some decrease in label incorporation into the N‐terminal coat protein region. According to the new ‘sandwich’ variant of our recently proposed model of the three‐dimensional structure of the intravirus PVX coat protein, the 176–198 segment is assigned to the β‐sheet region located at the subunit surface, presumably participating in coat protein interactions with the intravirus RNA and/or in protein–protein interactions, whereas the N‐terminal coat protein region corresponds to the other part of the same β‐sheet. For the remaining segments of the PVX coat protein subunit, no significant difference between tritium incorporation into untreated and TGBp1‐treated PVX was observed. A detailed description of the ‘sandwich’ version of the intravirus PVX coat protein model is presented.

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium

The structure of the C‐terminal domain of the influenza virus A matrix M1 protein, for which X‐ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally‐activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C‐terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C‐terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C‐terminal domain is an almost flat layer with a three‐α‐helical structure. To explain the high level of tritium label incorporation into the C‐terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI‐TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C‐terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell.

The In Situ Structural Characterization of the Influenza A Virus Matrix M1 Protein within a Virion

The first attempt has been made to suggest a model of influenza A virus matrix M1 protein spatial structure and molecule orientation within a virion on the basis of tritium planigraphy data and theoretical prediction results. Limited in situ proteolysis of the intact virions with bromelain and surface plasmon resonance spectroscopy study of the M1 protein interaction with lipid coated surfaces were used for independent confirmation of the proposed model.

Analysis of the role of the coat protein N-terminal segment in Potato virus X virion stability and functional activity

Previously, we have reported that intact Potato virus X (PVX) virions cannot be translated in cell-free systems, but acquire this capacity by the binding of PVX-specific triple gene block protein 1 (TGBp1) or after phosphorylation of the exposed N-terminal segment of intravirus coat protein (CP) by protein kinases. With the help of in vitro mutagenesis, a nonphosphorylatable PVX mutant (denoted ST PVX) was prepared in which all 12 S and T residues in the 20-residue-long N-terminal CP segment were substituted by A or G. Contrary to expectations, ST PVX was infectious, produced normal progeny and was translated in vitro in the absence of any additional factors. We suggest that the N-terminal PVX CP segment somehow participates in virion assembly in vivo and that CP subunits in ST virions may differ in structure from those in the wild-type (UK3 strain). In the present work, to test this suggestion, we performed a comparative tritium planigraphy study of CP structure in UK3 and ST virions. It was found that the profile of tritium incorporation into ST mutant virions in some CP segments differed from that of normal UK3 virions and from UK3 complexed with the PVX movement protein TGBp1. It is proposed that amino acid substitutions in ST CP and the TGBp1-driven remodelling of UK3 virions induce structural alterations in intravirus CPs. These alterations affect the predicted RNA recognition motif of PVX CP, but in different ways: for ST PVX, labelling is increased in α-helices 6 and 7, whereas, in remodelled UK3, labelling is increased in the β-sheet strands β3, β4 and β5.

Influenza virus hemagglutinin spike neck architectures and interaction with model enzymes evaluated by MALDI-TOF mass spectrometry and bioinformatics tools

Interactions between model enzymes and the influenza virus hemagglutinin (HA) homotrimeric spike were addressed. We digested influenza virions (naturally occurring strains and laboratory reassortants) with bromelain or subtilisin Carlsberg and analyzed by MALDI-TOF mass spectrometry the resulting HA2 C-terminal segments. All cleavage sites, together with (minor) sites detected in undigested HAs, were situated in the linker region that connects the transmembrane domain to the ectodomain. In addition to cleavage at highly favorable amino acids, various alternative enzyme preferences were found that strongly depended on the HA subtype/type. We also evaluated the surface electrostatic potentials, binding cleft topographies and spatial dimensions of stem bromelain (homologically modeled) and subtilisin Carlsberg (X-ray resolved). The results show that the enzymes (∼45Å(3)) would hardly fit into the small (∼18-20Å) linker region of the HA-spike. However, the HA membrane proximal ectodomain region was predicted to be intrinsically disordered. We propose that its motions allow steric adjustment of the enzymes active sites to the neck of the HA spike. The subtype/type-specific architectures in this region also influenced significantly the cleavage preferences of the enzymes.

Partially disordered structure in intravirus coat protein of potyvirus potato virus A.

Potyviruses represent the most biologically successful group of plant viruses, but to our knowledge, this work is the first detailed study of physicochemical characteristics of potyvirus virions. We measured the UV absorption, far and near UV circular dichroism spectra, intrinsic fluorescence spectra, and differential scanning calorimetry (DSC) melting curves of intact particles of a potato virus A (PVA). PVA virions proved to have a peculiar combination of physicochemical properties. The intravirus coat protein (CP) subunits were shown to contain an unusually high fraction of disordered structures, whereas PVA virions had an almost normal thermal stability. Upon heating from 20°C to 55°C, the fraction of disordered structures in the intravirus CP further increased, while PVA virions remained intact at up to 55°C, after which their disruption (and DSC melting) started. We suggest that the structure of PVA virions below 55°C is stabilized by interactions between the remaining structured segments of intravirus CP. It is not improbable that the biological efficiency of PVA relies on the disordered structure of intravirus CP.

Nonequilibrium processes in reactions of hot tritium atoms with cooled solid targets. Influence of the atomizer temperature on formation of labeled substances

A system consisting of a cold target and “hot” atoms generated by dissociation of tritium on a tungsten wire was studied with the aim to determine conditions for preparing tritium-labeled organic compounds with the maximal radiochemical yield. The influence of the atomizer temperature on the result of the reaction of tritium atoms with amino acids and tetraalkylammonium bromides was studied; homological series of the substrates were examined with the aim to evaluate the contributions of functional groups and hydrocarbon tail to the processes occurring in the target. The dependence of the yield of the labeled parent compound on the atomizer temperature varied in the range 1600–2000 K was determined. The rates of decarboxylation and deamination sharply grew with increasing temperature of the tungsten wire. The highest yield of labeled amino acids was attained at an atomizer temperature of 1800–1900 K, and at higher temperature their yield decreased. The difference between the activation energies of the elimination of the carboxy and amino groups and of the isotope exchange of hydrogen for tritium in the C-H bond appeared to be 93 and 59 kJ mol−1, respectively. For alkyltrimethylammonium bromides with the alkyl radicals C12H25, C14H29, and C16H33, the yield of the labeled parent compound reached 80–90% and was virtually independent of the atomizer temperature. The capability of tritium atoms to penetrate into the targets was evaluated. For the exponential model of the attenuation of the flow of tritium atoms inside the target, the attenuation factor for freeze-dried amino acids and alkyltrimethylammonium bromides as targets was 1.8 nm−1.