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Information × Registration Number 0218U001146, 0112U003747 , R & D reports Title Investigation of local comformation changes and metastable structural elements in tyrosyl-tRNA synthetases from prokaryotes and eukaryotes popup.stage_title Head Kornelyuk Alexander Ivanovich, Registration Date 24-01-2018 Organization Institute of Molecular Biology and Genetics of NASU popup.description2 Detailed investigation of the molecular dynamics of proteins of the eukaryotic and prokaryotic translation system: H. sapiens tyrosyl tRNA synthetase, B. taurus and M.tuberculosis, their mutant forms of substrate synthetase complexes, as well as the protein AIMP1 / p43, a component of the multisintetazic complex of eukaryotes. A computer simulation of the spatial structure of the full-size tyrosyl-tRNA synthetase of H. sapiens, its complexes with substrates and mutant forms associated with Sharko-Mari-Tus's neuropathy was performed. The investigation of the molecular dynamics of HsTyrRS showed the formation of dynamic elements of the secondary structure in the intermodule linker and in the unstructured catalytic loop. Modeling of the molecular dynamics of the mutant tyrosyl-tRNA synthetase forms associated with Sharko-Mari-Tus's neuropathy revealed the presence of local conformational changes in the unstructured loop of CP1-insert Roscman convolutions, which are accompanied by the formation of metastable b-structural elements (K147-E157). The analyzes of the trajectory of the molecular dynamics of the HsTyrRS * tRNAtyr complex showed the presence of hydrogen bonds, which confirm the presence of the amino acid residues of the CP1 loop insert in conjunction with homologous tRNATyr . Formation of metastable b-structural elements in this region can lead to violations in the recognition of the acceptor stem of the corresponding tRNA. A computer model of the HsTyrRS triple complex with tRNAtyr and eEF1A2 elongation factor was constructed. On the basis of this model, the mechanism of influence of Sharko-Mari-Tus mutations on the interaction with its partner macromolecules is proposed. It is suggested that the conformational effects detected may lead to changes in the conformation of the active center and to the change of the specific interactions of the HsTyrRS mutant forms with the eEF1A and / or RTATyr prolongation factor. The effect of changing the conformation of the remainder of His305 during the formation of the interface of recognition of the anticodonal stem tRNA and the importance of this conformation to maintain the stability of the complex was discovered. The molecular dynamics of TyrRS M.tuberculosis and the structural-functional changes of the catalytic KFGKS-loops, which are accompanied by the formation of two antiparallel? -pins, whose average life expectancy is 19-53 ps, is documented. The antiparallel b-pins formed in the catalytic loop support the loop in a closed M-conformation, directing the catalytic motif of KFGKS to the pocket of the active center and supporting the loop in the functional state ready for binding to the substrates. In the absence of b-structures in the catalytic loop, it emerges from the active center and goes into an open O-state. The M-conformation of the catalytic loop depends on the substrate in the active center. The loop adopts three different closed conformations: when the enzyme is in the free state (SCfree), the loop conformation when the active center is ATP (SCATP) and when the tyrosyl adenylate (SCYMP) is bound in the active center. The design of new inhibitors of the active center M.tuberculosis TyrRSSB-219383-a and SB-219383-b was carried out. Experimental studies of tyrosyl-tRNA-synthetase 39K fluorescence have been carried out, which indicates that the surfactant residues of tryptophan are available for fluorescence dries, but Trp40 is largely shielded in the molecule of the protein being studied and its fluorescence is quenched only with a neutral acrylamide dye. The quenching value of fluorescence indicates the diffusion of the stomach through the protein matrix due to confirmation fluctuations in the nanosecond time interval. A computer simulation of the spatial structure of the protein AIMP1 / p43 was carried out. The secondary structure of the AIMP1 / p43 protein, which corresponds to the secondary structure of the protein model we have obtained, is experimentally investigated by the method of circular dichroism, and the formation of a short fragment of ?-spiral in an unstructured protein region is detected. Investigation of the structure of AIMP1 / p43 by fluorescence spectroscopy indicates changes in the microprojection of the Trp271 residue in the process of local melting and the dynamic availability of Trp271 to solvent molecules due to conformational protein fluctuations in the solution. Computer simulation of the AIMP1 / p43 complex with tRNAPhe showed that tRNA binding occurs asymmetrically with two subunits of the protein, in particular, in the C-module with Lys124-Gly143 and Asn265-Gln273 regions forming a positively charged cluster. Formation of the ztRNA complex leads to the stabilization of the protein globuleAIMP1 / p43 due to simultaneous interaction with the N- and C-terminal modules AIMP1 / p43 and stabilization of the central unstructured protein portion. Stabilization of the structure of AIMP1 / p43 in combination with tRNA has been confirmed experimentally by fluorescence spectroscopy. Analysis of NMR spectra of AIMP1 / p43 protein was performed, which indicates the identity of the conformation of the C-terminal portion with the structure of the cytokineEMAP II. New signals are identified in the HSQC-1N / 15N spectrum of AIMP1 / p43, which confirm the formation of? -spiral fragments in the N-terminal portion of the polypeptide. Product Description popup.authors Болсунова О.І. Заїка Л.А. Задорожній Б.О. Карпова І.С. Кацан В.В. Коломієць Л.А Кордиш М.О. Корнелюк О.І. Лило В.В. Ложко Д.М. Потопальський А.І. Савицький О.В. popup.nrat_date 2020-04-02 Close