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石田 恒; 松本 淳
no journal, ,
To understand the mechanism of reverse tRNA translocation, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the pre-translocational states by fitting the complex into cryo-EM density maps. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The results indicate that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gatet. Conformational change of EF-G was interpreted as the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion.
河野 秀俊; 櫻庭 俊; 石田 恒
no journal, ,
Eukaryotic genome is compactly stored into a tiny nucleus of cell in the form of protein-DNA complex. This protein-DNA complex is called as nucleosome which is composed of a histone octamer formed by two copies each of the four core histones H3, H4, H2A and H2B and about 150 bp of DNA wrapping almost twice around the octamer. This compact form, however, has to be unwrapped in transcription, DNA duplication and DNA repair processes. To study the detailed molecular mechanism, free energy profiles for unwrapping nucleosomal DNA on several nucleosomes were calculated. So far, we estimated that a cost for unwrapping the outer DNA is 0.1 to 0.4 kcal/mol/1bp, consistent with a value experimentally obtained. In the meeting, we will report the cost for unwrapping the inner DNA.
角南 智子; 河野 秀俊
no journal, ,
Recently, zinc finger nucleases which are artificially fused a zincfinger transcription factor and a nuclease have been widely used for genome editing. However, the applications are limited because they only recognize GC-rich sequences. To make such artificial proteins recognize broader sequences other than GC-rich ones, we are rationally designing novel proteins based on an engrailed homeodomain. Engrailed homeodomain proteins recognize six bases. To recognize longer DNA sequences, we connected two homeodomains with a short linker. We will report that the designed molecule show a strong affinity and high specificity for the target sequence. We will also report a DNA sequence profile of the protein obtained by B1H assay.
松本 淳; 高木 淳一*; 岩崎 憲治*
no journal, ,
We have been developing a new computational approach to build a 3D atomic model from an electron microscope (EM) image of a biological molecule. In the usual approach, a 3D-EM model is constructed from many EM images. Then, a 3D atomic model is built mainly by deforming the X-ray crystal structure so that it fits into the 3D-EM model better. Our approach, on the other hand, uses only a single EM image (or an averaged image) and an X-ray crystal structure (or a modeled structure), and has the advantage of being applicable even to flexible molecules, for which it is difficult to construct 3D-EM models. We will report the results about the application to integrins, transmembrane proteins that involved in cell-cell and cell-extracellular matrix adhesions.
松尾 龍人; 荒田 敏昭*; 小田 俊郎*; 藤原 悟
no journal, ,
中性子準弾性散乱を用いて、F-アクチン及びミオシンS1タンパク質のダイナミクスを調べた。J-PARCのAMATERASにおいて、F-アクチン及びミオシンS1の溶液試料を用いて実験を行った。ミオシンS1構成原子の運動の相関時間は、ヘモグロビン等他のタンパク質と同程度であったが、F-アクチンの相関時間はS1よりも小さいことが分かった。また、F-アクチン構成原子はS1よりも大きな空間を揺らぐことが分かった。これらの結果は、F-アクチンがミオシンS1よりも柔軟であることを示唆している。
-synuclein detected by neutron scattering藤原 悟; 荒木 克哉*; 松尾 龍人; 八木 寿梓*; 山田 武*; 柴田 薫; 望月 秀樹*
no journal, ,
The protein, -synuclein (-Syn) forms amyloid fibrils. Formation of amyloid fibrils is associated with the pathogenesis of a neuro-degenerative disorder, Parkinson's disease. In order to obtain insights into the role of the protein dynamics in the mechanism of amyloid fibril formation, we carried out quasielastic neutron scattering experiments and characterized the "dynamic" behavior of -Syn. The measurements on the solution samples of -Syn in the monomeric and fibril states were carried out using a high energy resolution near-backscattering spectrometer, BL02 (DNA), at MLF/J-PARC, Japan. Differences in the dynamical behavior of the protein were detected between the monomeric and fibril states. Analysis of the spectra obtained suggested an entropy-driven mechanism of amyloid fibril formation.