Crystallization and X-ray crystallographic analysis of Complexes III and I in mitochondrial respiratory system

线粒体呼吸系统中复合物 III 和 I 的结晶和 X 射线晶体分析

• 批准号: 03454565
• 负责人: YOSHIKAWA Shinya
• 金额: $ 3.39万
• 依托单位: Himeji Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454565/
• 关键词: 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

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.

本文在总结了牛心细胞色素c氧化酶结晶的基础上，对Rieske等报道的牛心复合物Ⅲ的纯化工艺进行了改进，提高了产率和重现性。用非离子洗涤剂十二烷基蔗糖完全取代纯化样品中的残留胆酸盐，显著增加了样品的稳定性。溶解在Tris-HCl缓冲液中的制剂可在聚乙二醇作为沉淀剂的情况下以单斜晶形式结晶，得到具有7.5 A分辨率的X射线衍射图。但是，晶体易碎，容易孪生。在K-磷酸盐缓冲液中的制备提供了六方晶体，其衍射X射线高达6.5 A分辨率。An^<2+>的加入提高了六方晶体结晶的重现性。另一方面，用结晶纯化的制备物得到四氢呋喃晶体，其结构类似于六方晶体。还重新检查了Rieske对复合物I的纯化程序，以确定重现性和收率（从1.2kg牛肉心肌中提取0.9g）。用“结晶筛”（汉普顿研究公司）调查结晶条件。提供了在偏振光下显示出闪烁的球粒晶体。

项目成果

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.

Kawamoto, M.、Kubota, T.、Matsunaga, T.、Fukuyama, K.、Matsubara, H.、Shinzawa-Itoh, K. 和 Yoshikawa, S.：“新晶型和初步 X 射线衍射研究

Kubota, T., Yoshikawa, S.and Matsubara, H.: "Kinetic mechanism of beef heart ubiquinol : cytochrome c oxido-reductase." J.Biochem.111. 91-98 (1992)

Kubota, T.、Yoshikawa, S. 和 Matsubara, H.：“牛心泛醇的动力学机制：细胞色素 C 氧化还原酶。”

Yoshikawa,S.: "Strategies for crystallization of membrane protein complexes." J.Cryst.Growth. 122. 298-302 (1992)

Yoshikawa,S.：“膜蛋白复合物的结晶策略。”

Kubota,T.: "Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase." J.Biochem.111. 91-98 (1992)

Kubota,T.：“牛心泛醇的动力学机制：细胞色素 C 氧化还原酶。”

Kawamoto,M.: "New crystal forms and preliminary X-ray diffraction studies of mitochondrial cytochrome bc_1 complex from bovine heart." J.Mol.Biol.(submitted).

Kawamoto,M.：“牛心脏线粒体细胞色素 bc_1 复合物的新晶体形式和初步 X 射线衍射研究。”