| Project/Area Number |
01304061
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| Research Category |
Grant-in-Aid for Co-operative Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | The Institute of Physical and Chemical Research |
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Principal Investigator |
KOUYAMA Tsutomu Inst. of Phys. & Chem. Research, Biophysics Lab., Research Scientist, 生物物理学研究室, 研究員 (30170210)
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| Co-Investigator(Kenkyū-buntansha) |
MIKI Kunio Tokyo Univ. of Technol, Asoc. Proffesor, 資源化学研究所, 助教授 (10116105)
SHIRAKIHARA Yasuo Hyogo Univ. of Education, Div. Science, Assoce. Professor, 自然系物理, 助教授 (20150287)
IWASA Tatsuo Himeji Univ. of Technol., Fac. Science, Assoc. Professor, 理学部, 助教授 (00133926)
TOMIOKA Akihiro Univ. of Tokyo. Fac. Engineering, Asistant Professor, 工学部, 助手 (10211400)
KUSUMI Akihiro Univ. of Tokyo., College of Arts and Science, Assoc. Professor, 教養部, 助教授 (50169992)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥10,800,000 (Direct Cost: ¥10,800,000)
Fiscal Year 1990: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 1989: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Keywords | Membrane Protein, / Crystallization, / Detergent, / Protein Crystallography, / Bacteriorhodopsin, / Chytochrome C oxidase, / F1-ATPase, / Photosynthetic Reaction Center / 光反応中心 / F1ーATPase / べん毛モ-タ- / 生体膜 / 蛋白質の結晶化 / ロドプシン・ファミリ- / H^+-ATPase / Ca^<++>結合膜蛋白質 / 遺伝子組換え蛋白質 |
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| Research Abstract |
Protein crystallization is a bottle-necked process in crystallographic analyses of the tertiary structure of proteins. Especially membrane proteins are very difficult to crystallize. In this research project, we challenged to crystallize many membrane proteins, aiming at establishing general rules for the three-dimensional crystallization of membrane proteins. The membrane proteins that we examined are : bacteriorhodopsin, rhodopsin (bovine and octopus), receptor proteins of neurotransmitters, cytochrome c oxidase (bovine heart), photosynthetic reaction center (a red alga), Ca++-binding membrane proteins, the Mling protein of flagellar motors (Salmonella), H+-ATPase (a thermophilic bacterium PS3). Actin and recA protein, which are known to self-assemble to form filamentary aggregates, were also investigated. Difficulty in making high-quality crystals of membrane proteins or hydrophobic pfoteins comes primarily from their instability in solubilized or monomeric states. Thus we tried to
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understand the physical forces stabilizing the tertiary structure of membrane proteins ; e. g., protein denaturation and protein-protein interaction were investigated in the presence of man of detergents and chemicals (e. g., alcohols).
The project team consisted of young biophysicists who had been long working on membrane proteins. But only a few members had been familiar to the protein crystallography. In order to enlighten and encourage each other, therefore, we hold several public meetings and discussed the difficulties that were encountered in the trials of protein crystallization. We were surprised to find a large audience in the meetings, because we had thought that the protein crystallography was not very active in Japan. Now we understand that a large number of researchers are interested to protein crystallization. We are convinced that the protein crystallography in Japan will become very active in future.
In the discussion about crystallization techniques, the followings were proposed to be prominent : 1) A high-quality crystal can be grown by decreasing or increasing temperature very slowly, when the protein solubility is significantly temperature-dependent ; 2) Two kinds of detergent can be used in combination, when one of them is too strong to keep the protein structure while the other is too mild to solubilize membranes ; 3) Vapor diffusion in the presence of a temperature gradient allows proteins to be concentrated locally and thus to be crystallized in a restricted region. Also, many other factors that influence protein crystallization, i. e., the gravity, the purity of protein, the self-oxidation of protein, were discussed. Video-recording of crystallization processes was proven to be powerful in understanding the mechanism of protein-protein association. Less
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