Crystallization and X-ray crystallographic analysis of Complexes III and I in mitochondrial respiratory system
| Project/Area Number |
03454565
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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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 | Himeji Institute of Technology |
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Principal Investigator |
YOSHIKAWA Shinya Himeji Inst. of Tech. Prof., 理学部, 教授 (40068119)
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| Co-Investigator(Kenkyū-buntansha) |
TSUKIHARA Tomitake Tokushima Univ. Prof., 工学部, 教授 (00032277)
SHINZAWA-ITOH Kyoko Himeji Inst. of Tech. Asis. Prof., 理学部, 助手 (70206316)
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| Project Period (FY) |
1990 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1993: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1992: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1991: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | NADH-Ubiquinone reductase / Ubiquinol-cytochrome c reductase / Membrane protein / Electron transfer system / respiratory chain / Protein crystallizatioin / Crystallographic analysis / 複合体I / チトクロムbC_1,複合体 |
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| Research Abstract |
Based on the experience about crystallization of beef heart cytochrome c oxidase, purification procedure of beef heart Complex III, reported by Rieske, was improved to increase the yeald and the reproducibility. Complete replacement of the residual cholate in the purified sample with a nonionic detergent, dodecyl sucrose, increased the stability of the sample significantly. The preparation dissolved in Tris-HCl buffer was crystallizable in a monoclinic form with polyethyleneglycol as the precipitant, giving an x-ray diffraction pattern with 7.5 A resolution. However, the crystals were fragil and easily to be twinned. The preparation in K-phosphate buffer provided hexagonal crystals which diffracted x-rays upto 6.5 A resolution. An addition of An^<2+> increased the reproducibility of crystallization of the hexagonal crystals. On the other hand, the preparation purified with crystallization gave tetragonal crystals with a parfection similar to the hexagonal one.
Purification procedure by Rieske for Complex I was also reexamined to establish the reproducibility and the yeald(0.9 grams from 1.2 kg of beef heart muscle). Survey of the crystallization conditions with "crystal screen"(Hampton Research Inc.) provided spherlite crystals which showed birefringe under polarized light.
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Report
(4 results)
Research Products
(11 results)
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[Publications] Yoshikawa, S., Shinzawa-Itoh, K., Ueda, H., Tsukihara, T., Fukumoto, Y., Kubota, T., Kawamoto, M., Fukuyama, K.and Matsubara, H.: "Strategies for crystallization of membrane protein complexes." J.Cryst. Growth. 122. 298-302 (1992)
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[Publications] Kawamoto, M., Kubota, T., Matsunaga, T., Fukuyama, K., Matsubara, H., Shinzawa-Itoh, K., and Yoshikawa, S.: "New crystal form and preliminary X-ray diffraction studies of mitochondrial cytochrome bc_1 comples from bovine heart." submitted to J.Mol. Biol.
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