Folding and crystallization conditions of menbrane protein supercomplexes

膜蛋白超复合物的折叠和结晶条件

• 批准号: 03304056
• 负责人: YOSHIKAWA Shinya
• 金额: $ 10.88万
• 依托单位: Himeji Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Co-operative Research (A)
• 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/grant/KAKENHI-PROJECT-03304056/
• 关键词: Membrane protein; Supermolecular complex; X-ray crystallographic analysis; Electron micrographic analysis; Folding mechanism; Crystal growth mechanism; Crystallization; Three dimensional-stractural prediction; 結晶構造解析; チトクロム酸化酵素; チトクロムbc_1複合体

This research project has provided the following conclusion about the folding mechanism and the crystallization method of membrane protein. Three dimensional structure of the membrane protein supercomplexe spanned across the biological membrane in the cell is stabilized by hydrophilic loops exposed to aquaous medium as well as by alpha-helices placed under the ydrophobic environment in the membrane which provide hydrogen bonds and electrostatic interactions between these helices. Thus, solubilization with a detergent which covers the hydrophobic surface seems the best method for isolation of this type of protein complex without denaturation. The specific interaction between the protein melocules which stabilizes the crystalline state is likely to be the one between the hydrophilic surfaces, suggesting that larger homologous membrane protein is better for crystallization than the smaller one. This conclusion is suppoted by the fact that only the biggest cytochrome c oxidase and cytochrome bc1 complex, isolated from beef heart mitochondria, among many homologous proteins have been crystallized. Structure of the detergent molecules which attach to the hydrophobic surfaces of membrane proteins could control the specific interactions between the hydrophilic surface of the protein moleules to determine the crystallization conditions. The control mechanism is not so simple as has been proposed by Michel et al.

本研究项目对膜蛋白的折叠机制和结晶方法提供了以下结论。跨越细胞生物膜的膜蛋白超复合物的三维结构通过暴露于水介质的亲水环以及膜中疏水环境下的α螺旋来稳定，α螺旋在这些螺旋之间提供氢键和静电相互作用。因此，用覆盖疏水表面的去污剂增溶似乎是分离此类蛋白质复合物而不变性的最佳方法。稳定结晶状态的蛋白质分子之间的特异性相互作用很可能是亲水表面之间的相互作用，这表明较大的同源膜蛋白比较小的同源膜蛋白更适合结晶。这一结论是由以下事实支持的：在众多同源蛋白中，只有从牛心线粒体中分离出的最大的细胞色素c氧化酶和细胞色素bc1复合物已结晶。附着在膜蛋白疏水表面的去污剂分子的结构可以控制蛋白质分子亲水表面之间的特定相互作用，从而确定结晶条件。控制机制并不像 Michel 等人提出的那么简单。

项目成果

H.Nakayama: "Photolabeled sites with a tetrodotoxin derivative in the domain III and IV of the electroplax sodium channel." Biochem.Biophys.Res.Commun. 184. 900-907 (1992)

H.Nakayama：“在 electroplax 钠通道的 III 域和 IV 域中用河豚毒素衍生物进行光标记的位点。”

三木邦夫: "蛋白工学とコンピューター蛋白質のX線結晶構造解析" CICSJ Bulletin. 11(4). 29-32 (1993)

Kunio Miki：“蛋白质工程和蛋白质的计算 X 射线晶体结构分析”CICSJ 公告 11(4) (1993)。

三木邦夫: "タンパク質結晶構造解析総括プログラムシステム,“PROTEIN"の現況" 日本結晶学会誌. 34. 365-366 (1992)

Kunio Miki：“蛋白质晶体结构分析综合程序系统的现状，‘PROTEIN’”日本晶体学会杂志 34. 365-366 (1992)。

K.Fukuda,T.Kouyama.: "Photoreaction of bacteriorhodopsin at high pH:Origins of the slow decay components of M." Biochemistry. 31. 11740-11747 (1992)

K.Fukuda,T.Kouyama.：“细菌视紫红质在高 pH 下的光反应：M 缓慢衰变成分的起源。”

T.Kouyama.: "Bacteriorhodopsin,a bilogical proton pump working in the light." RIKEN Review. 1. 5-6 (1993)

T.Kouyama：“细菌视紫红质，一种在光下工作的生物质子泵。”