成果報告書詳細
管理番号20100000001561
タイトル*平成21年度中間年報 創薬加速に向けたタンパク質構造解析基盤技術開発 創薬加速に向けたタンパク質構造解析基盤技術開発
公開日2011/4/20
報告書年度2009 - 2009
委託先名社団法人バイオ産業情報化コンソーシアム
プロジェクト番号P08005
部署名バイオテクノロジー・医療技術開発部
和文要約和文要約等以下本編抜粋:1.研究開発の内容及び成果等
(1) 研究開発項目1「「電子線等による膜タンパク質及びその複合体の構造解析技術」
(1~1) 膜タンパク質及びその複合体の構造解析に必要な膜タンパク質等の発現・精製技術、
結晶化技術の開発
〔バイオメディシナル情報研究センター/京都大学分室/京都大学大学院理学研究科〕
(1~1~1)組み換え遺伝子技術と昆虫細胞等による発現系を用いて、解析が求められているヒト等真核生物由来の膜タンパク質の発現・精製法の開発を行ってきた。特に水チャネル、イオンチャネル、GPCRなど、創薬分野から期待されている膜タンパク質の構造解析を目指して大量発現・精製の研究を進めた。具体的には、脳に発現する水チャネル、AQP4とその変異体、ギャップ結合チャネルCx26とその変異体、エンドセリン受容体、特にETBRとそのキメラや変異
体の昆虫細胞を用いた発現と精製が図1に示す様に安定に行えるような技術を確立した。また、GPCRとHomerを用いて界面活性剤による可溶化なしに発現・精製できる技術の開発を進めた。
英文要約Title:Structural Guided Drug Development FY2009 Annual Report
The structure of a mutant aquaporin-4 mimicking phosphorylation of Ser180, which has been implicated in the regulation of AQP4 water permeability by dopamine, was analysed at 2.8 ~ resolution and the analysis enabled us to observe eight water molecules in the channel. The quality of the map of water molecules in our analysis is far better than the map by X-ray crystallography at higher resolution. By our analysis, we could prove the H-bond isolation mechanism. Two papers, in which we discussed function of ion channels, were published. We proposed plug gating mechanism based on structure analyses of gap junction channel of Cx26. By improving computer program for multilayered 2D-crystals, we could improve quality of structure analyses of AQP4, Cx26 and H+,K+-ATPase. Structure of erythrocyte band 3 was analysed by iterative helical real-space reconstruction. Crystal structure of CIA in complex with a domain of the TFIID complex was analysed by X-ray crystallography.
For NMR structural determinations of proteins, an aggregation of target proteins is one of the major obstacles. To overcome the limitation, we established a rapid searching method to optimize NMR sample solution, by fluorescence correlation spectroscopy (FCS).
Kluyveromyces lactis is superior to methylotrophic yeast P. pastoris in some respects; easy and fast transformation, good reproducibility of induction of protein expression and so on. By utilizing these advances of K. lactis, we established noble protein expression systems, which enable us to prepare labeled proteins by using glucose and ammonium chloride as stable isotope source.
In collaboration with Prof. Nakamura’s team in the project, we developed a new modeling of protein complexes based upon amino acid-selective cross-saturation (ASCS) method. We also investigated the molecular recognition of drag target proteins, such as a chemokine receptor, CXCR4, a discoidin receptor, DDR2, and cell surface receptors, CD44 and GPIV.
Prediction of protein-drug complex structures and their affinities by computation, called as in-silico drug docking, is one of the core technologies for structure-guided drug development. So far, we have developed our own in-silico docking program suite, "myPresto", and have released it to public. For finding non-peptide ligands with scaffold hopping from peptide ligands, Molecular Dynamics Maximum Volume Overlap (MD-MVO) method has been developed. A new fragment screening by replica generation (FSRG) has been developed to overcome the problem that the fragments are generally too small, by linking artificial side chains to fragments to enhance its size and affinity. Aqueous solubility of any druggable chemical compound is also predicted with high accuracy. Those technologies have been applied to many receptor proteins, mainly to membrane proteins, such as human ~-opioid receptor and hERG channel, and satisfactory predictions for inhibition by particular compounds have been attained.
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