[Differences in spatial structures of the influenza virus M1 protein in crystal, solution and virion].

Spatial structure of the influenza virus A/Puerto Rico/8/34 (PR8, subtype H1N1) M1 protein in a solution and composition of the virion was studied by tritium planigraphy technique. The special algorithm for modeling of the spatial structure is used to simulate the experiment, as well as a set of algorithms predicting secondary structure and disordered regions in proteins. Tertiary structures were refined using the program Rosetta. To compare the structures in solution and in virion, also used the X-ray diffraction data for NM-domain. The main difference between protein structure in solution and crystal is observed in the contact region of N- and M-domains, which are more densely packed in the crystalline state. Locations include the maximum label is almost identical to the unstructured regions of proteins predicted by bioinformatics analysis. These areas are concentrated in the C-domain and in the loop regions between the M-, N-, and C-domains. Analytical centrifugation and dynamic laser light scattering confirm data of tritium planigraphy. Anomalous hydrodynamic size, and low structuring of the M1 protein in solution were found. The multifunctionality of protein in the cell appears to be associated with its plastic tertiary structure, which provides at the expense of unstructured regions of contact with various molecules-partners.

[Disordered regions in C-domain structure of influenza virus M1 protein].

Influenza virus matrix M1 protein is one of the main structural components of the virion performing also many different functions in infected cell. X-ray analysis data with 2.08 angstrom resolution were obtained only for the N-terminal part of M1 protein molecule (residues 2-158) but not for its C-terminal domain (159-252). In the present work M1 protein of A/Puerto Rico/8/34 (H1N1) virus strain in acidic solution was investigated with the help of tritium bombardment. Tritium label incorporation into M1 protein domains preferentially labeled the C-domain and inter-domain loops. Analytical centrifugation and dynamic light scattering experiments demonstrated increased hydrodynamic parameters (diameter) that may be explained by low degree of M1 structural organization. Computational analysis of M1 protein by intrinsic disorder predictions methods also demonstrated the presence of unfolded regions mostly in the C-domain and inter-domain loops. It is suggested, that influenza virus M1 polyfunctionality in infected cell is determined by its tertiary structure plasticity which in its turn results from the presence of unstructured regions.

[Modeling of protein spatial structure using tritium planigraphy].

The results of proteins spatial structure modeling using the tritium planigraphy technique are presented. The knowledge of three-dimensional structure of macromolecules is extremely necessary to understand the basic mechanisms of interaction in biological systems and complex technological processes. Known limitations of the X-ray analysis (crystal state) and NMR (molecular weight) make it necessary to seek new approaches to modeling the spatial structure of proteins. Semiempirical tritium planigraphy technique is one of these approaches. The method is based on the bombardment of the object by beam of hot tritium atoms (E(at) > or = 0.3 eV) and a computer simulation experiment. On the example of proteins of the different structural classes we set that by using this integrated approach can be obtained by three-dimensional model of the structure, well consistent with the data of X-ray analysis. An important factor is a sequence search of contacts between secondary structure elements: the best fit model with the native structure is achieved by assembling the elements of a vector in the sequence from the N- to C-terminus of the polypeptide chain.

Studying the spatial organization of membrane proteins by means of tritium stratigraphy: bacteriorhodopsin in purple membrane.

The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10-29 of helix A. They turned out to be low (0.04+/-0.02 A(-1)) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15+/-0.05 A(-1)) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane.

Tritium planigraphy: from the accessible surface to the spatial structure of a protein.

The method of tritium planigraphy, which provides comprehensive information on the accessible surface of macromolecules, allows an attempt at reconstructing the three-dimensional structure of a protein from the experimental data on residue accessibility for labeling. The semiempirical algorithm proposed for globular proteins involves (i) predicting theoretically the secondary structure elements (SSEs), (ii) experimentally determining the residue-accessibility profile by bombarding the whole protein with a beam of hot tritium atoms, (iii) generating the residue-accessibility profiles for isolated SSEs by computer simulation, (iv) locating the contacts between SSEs by collating the experimental and simulated accessibility profiles, and (v) assembling the SSEs into a compact model via these contact regions in accordance with certain rules. For sperm whale myoglobin, carp and pike parvalbumins, the lambda cro repressor, and hen egg lysozyme, this algorithm yields the most realistic models when SSEs are assembled sequentially from the amino to the carboxyl end of the protein chain.

[The concept of the "accessible surface" of the protein within the framework of tritium planigraphy. Experiment and calculation]. / Poniatie "dostupnaia poverkhnost" belka v ramkakh metoda tritevoi planigrafii. Eksperiment i raschet.

Tritium planigraphy allows one to determine the sterical accessibility of protein hydrocarbon fragments (CH, CH2, CH3 groups) for interaction with tritium atoms on condition of direct transit of the bombarding particles. Using lysozyme as a test system, it was shown to be possible to use these data for the description of protein accessible surface area in terms defined by Lee and Richards. The best agreement between experimental and theoretical results was achieved for an effective radius of the testing probe of 0.9 A. Coefficients were obtained that allowed calculation of the accessibility of amino acid residue as a whole using data about its hydrocarbon fragment accessibility.

[Accessible surface and intramolecular mobility of proteins: study by method of tritium planigraphy]. / Dostupnaia poverkhnost' i vnutrimolekuliarnaia podvizhnost' belkov: issledovanie metodom tritievoi planigrafii.

The method of tritium planigraphy is used for determination of the accessible surface of a globular protein--lysozyme--and the accessibility of particular types of amino acid residues as a function of temperature in the range of 77-193 K. Protein powder with humidity 10 +/- 1% was used. As the temperature is changed from 77 to 160 K for all types of amino acid residues was obtained decreasing of inclusion of tritium label. All types of amino acid residues may be divided on the three groups: I. Accessibility rises under increasing temperature from 160 to 293 K (K, R, H, P, L); II. Accessibility not depends or slightly increases with the growth of temperature (C, V, A, I, Y, F); III. Accessibility strongly increases in the range of the temperature 260-293 K (S, T, G, D + N, E + Q). The reason of "cold denaturation" is perhaps the difference of behaviour of structural water molecules. Under increasing of temperature from 160 to 293 K change of accessibility was explained the growing of intramolecular flexibility of molecule. Under transition from 160 to 77 K for all types of residues is observed sensible change of spatial structure of the protein, which cannot be explained by only participation of dynamical characteristics.

[Quantitative determination of the accessible surface of globular proteins by tritium planigraphy]. / Kolichestvennoe opredelenie dostupnoi poverkhnosti globuliarnykh belkov metodom tritievoi planigrafii.

At the last time the term "accessible surface" is used for a description of protein structure. The tritium planigraphy was used for quantitative detection of the accessible surface. The experimental dependence of an interaction probability of the tritium atoms with globular proteins with calculated accessible surface areas was obtained. The method was proposed on the basis of the information about the reactivity of amino acids residues and was used for the determination of an accessible surface of parvalbumin III of pike.