E. N. Bogacheva

Intrinsically unstructured regions in the C domain of the influenza virus M1 protein

The M1 matrix protein of the influenza virus is one of the main structural components of the virion that performs several different functions in the infected cell. X-ray analysis (with 2.08 Å resolution) has been performed for the N-terminal part of the M1 protein (residues 2–158) but not for its C-terminal domain (159–252). In the present study, we analyzed the structure of the M1 protein of the influenza virus A/Puerto Rico/8/34 (H1N1) strain in acidic solution using tritium planigraphy. The incorporation of tritium label into the domains of the M1 protein were studied; the C domain and the interdomain loops are preferentially accessible to tritium. Analytical centrifugation and dynamic laser light scattering demonstrated anomalous hydrodynamic parameters and low structuredness of the M1 protein, which has also been confirmed by circular dichroism data. Bioinformatic analysis of the M1 protein sequence revealed intrinsically unstructured segments that were concentrated in the C domain and interdomain loops between the N-, M-, and C domains. We suggest that the multifunctionality of the M1 protein in a cell is determined by the plasticity of its tertiary structure, which is caused by the presence of intrinsically unstructured segments.

Tritium planigraphy: A tool for studying the spatial structure of proteins and their complexes

The review is devoted to tritium planigraphy and its applications in solving a broad scope of problems in modern molecular and physicochemical biology. The method is based on nonselective substitution of tritium for hydrogen in the hydrocarbon parts of target molecules. It furnishes information on the steric accessibility of the components of a system under study (macromolecule within a complex amino acid residues, and even separate atomic groupings in a macromolecule) that characterizes the structure of the entire object. The technique is applicable to specimens in different phase states and has no limitations in respect of the target molecular mass. Tritium planigraphy is especially important in the cases when the biological macromolecules cannot be examined by the conventional methods (X-ray analysis and NMR spectroscopy). The review summarizes the studies of protein accessible surface and spatial arrangement, and outlines the approaches to modeling the protein 3D structure and probing into the spatial organization of theEscherichia coli ribosome and virus particles.

Tritium planigraphy as a tool for studying the structural organization nanobiocomplexes

The tritium planigraphy method is based on the nonselective substitution of radioactive isotope tritium for hydrogen in hydrocarbon fragments of molecules by means of a chemical reaction involving hot tritium atoms. Data on the steric accessibility of the system components (macromolecules in the complex, amino acid residues, and even individual atomic groups of macromolecules) characterize the structure of the object. The method, applicable to substances in different phase states, has no restrictions on the molecular weight of the target. Tritium planigraphy, used equally successfully in both crystals and solutions, makes it possible to study fine changes in the structure. The main results of studies of the structure of nanosized biocompexes by tritium planigraphy are presented.

Tritium planigraphy and nanosized biological particles

Advances in studies of protein and complex biological systems by tritium planigraphy are considered. Particular attention is paid to detailing the structural organization of the protein components of the influenza virus. The structure of the M1 protein in a solution, virion, and crystal is analyzed. The specific features of this protein identified using tritium planigraphy and computer simulation are indicative of significant changes in its conformation depending on the “phase” state. Models of the spatial structure of the M1 protein in solution and in the virion are proposed. Structural disorder is observed in one of the three domains of this protein. Ideas on the importance of this structure for the multifunctional properties of the protein, such as binding to the membrane and a ribonucleoprotein complex, as well as the transfer of genetic material during viral infection of healthy cells, were suggested.

Tritium planigraphy: Differences in the spatial structures of the influenza virus M1 protein in crystal, solution, and virion

The structure of the M1 protein of the influenza virus A/Puerto Rico/8/34 (PR8, subtype H1N1) in solution at acidic pH and in the composition of the virion has been studied by the tritium planigraphy method. A model of the spatial structure was constructed using a special algorithm simulating the experiment and a set of algorithms for predicting the secondary structure and disordered regions in proteins. The tertiary structure was refined using the Rosetta program. For a comparison of the structures in solution and inside the virion, the data of X-ray diffraction analysis for the NM domain were also used. The main difference in the structures of the protein in solution and the crystalline state is observed in the region of contact of N and M domains, which in the crystalline state is packed more densely. The regions of the maximum label incorporation almost completely coincide with unstructured regions in the protein that were predicted by the bioinformatics analysis. These regions are concentrated in the C domain and in loop regions between M, N, and C domains. The data were confirmed by analytical centrifugation and dynamic light scattering. Anomalous hydrodynamic dimensions and a low structuration of the M1 protein in solution were found. The polyfunctionality of the protein in the cell is probably related to its flexible tertiary structure, which, owing to unstructured regions, provides contact with various partner molecules.