A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological рН value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development.

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.

Рассмотрены достижения метода тритиевой планиграфии при исследовании белков и сложных биологических комплексов. Особое внимание уделено детализации структурной организации белковых компонентов вируса гриппа. Проанализирована структура белка М1 в растворе, в составе вириона и в кристалле. Выявленные с помощью метода тритиевой планиграфии и компьютерного моделирования особенности этого белка говорят о его значительном конформационном изменении при изменении “фазового” состояния. Предложены модели пространственной структуры белка М1 в растворе и вирионе. Обнаружена неупорядоченность одного из трех доменов этого белка. Высказаны предположения о важности такой структуры для полифункциональных свойств белка: связывания с мембраной, рибонуклеопротеиновым комплексом и переноса генетического материала в процессе инфицирования вирусом здоровой клетки.

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.

Методом тритиевой планиграфии исследована структура белка М1 вируса гриппа А/Puerto Rico/8/34 (PR8, подтип H1N1) в растворе при кислом рН и в составе вириона. Для построения моделей пространственной структуры использован специальный алгоритм, имитирующий эксперимент, а также набор алгоритмов предсказания вторичной структуры и неупорядоченных областей в белках. Третичную структуру уточняли при помощи программы Rosetta. Чтобы сравнить структуры в растворе и вирионе, использовали также данные рентгеноструктурного анализа для NM-домена. Основное различие структур белка в растворе и в кристаллическом состоянии наблюдается в области контакта N- и М-доменов, которая в кристаллическом состоянии упакована более плотно. Места максимального включения метки практически совпадают с неструктурированными областями в белке, предсказанными с помощью биоинформатического анализа. Эти области сконцентрированы в С-домене и в петлевых областях между М-, N- и С-доменами. Данные подтверждены с помощью аналитического центрифугирования и динамического светорассеяния. Обнаружены аномальные гидродинамические размеры и низкая структурированность М1-белка в растворе. Полифункциональность белка в клетке, по-видимому, связана с его пластичной третичной структурой, которая обеспечивает за счет неструктурированных областей контакт с различными молекулами-партнерами.

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.

Матриксный белок М1 вируса гриппа – один из основных структурных компонентов вириона, выполняющий в ходе вирусной инфекции ряд различных функций. Данные рентгеноструктурного анализа (с разрешением 2.08 A) существуют лишь для N-концевой части этого белка (остатки 2–158), не содержащей С-концевой домен (159–252). Структуру белка М1 вируса гриппа A/Puerto Rico/8/34 (H1N1) в кислой среде исследовали методом тритиевой планиграфии. Проанализировано включение тритиевой метки в домены белка М1 и установлена преимущественная доступность тритию С-домена и петлевых междоменных областей. С помощью аналитического центрифугирования и динамического лазерного светорассеяния обнаружены аномальные гидродинамические размеры и низкая структурированность белка М1, подтвержденная также данными кругового дихроизма. Биоинформатический анализ последовательности белка М1 выявил наличие неструктурированных областей, сконцентрированных в С-домене и в петлевых областях между N, М и С-доменами. Высказано предположение о том, что полифункциональность белка в клетке обусловлена пластичностью его третичной структуры, возникающей за счет неструктурированных участков.

The results of protein spatial structure modeling using the tritium planigraphy technique are presented. The knowledge of 3D structure of macromolecules is obligatory for understanding 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 is one of these approaches. The method is based on bombardment of the object with a beam of hot tritium atoms ( E at ≥ 0.3 eV) and computer simulation. On the example of proteins of different structural classes, we show that this integrated approach can yield a 3D model well consistent with the X-ray data. An important factor is the sequence of searching for contacts between secondary structure elements: the best fit with the native structure is achieved by assembling the elements from the N- to the C-terminus of the polypeptide chain.

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.

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.