Structural study of membrane proteins (GPCR and channels) regulating signal transduction.

从2001年起，在格兰特特别促进研究援助的支持下，在三年的时间里取得了以下研究成果。1)采用Sf9细胞过表达技术和电子结晶学方法分析水通道蛋白AQP4的结构。基于这种结构，我们可以解释水通道分子的正交阵形成和细胞黏附的机理。2)用冷冻电子显微镜分析乙酰胆碱受体孔的结构和门控机制(J.Mol.Biol，319,1165-1176(2002)，Natural，423,949-955(2003))。3)开发了用于分析电压敏感钠通道结构的单粒子分析自动拾取方法(Natural，409,1047-1051(2001))和分析IP_3受体结构的计算机程序(J.Struct.Biol，136,227-238(2001))。4)用X射线结晶学(J.Mol.Biol，318,1117-1126(2002))分析了Hmer的结构，并发展了一种新的共定位表达技术(Co-LET)，用于无洗涤剂纯化膜蛋白(BBRC，295,756-765(2002))。5)我们研究了内皮素B受体与Caveoline-1的相互作用(Eur.J.Bioch.，270,1816-1827(2003))。视紫红质的结构分析在2.6Å(Proc.Natl.Academy.Science USA，99,5982-5987(2002))。研究了连接蛋白26中三个残基的突变，以获得该分子结构的洞察力(J.Boil.Chem.，278,1807-1816(2003))。第二个PDZ结构域对PSD-95聚集的重要性被揭示(J.Biol.Chem.，277,3640-3646(2002))。

The following research results were achieved in three years by support of Glant-in Aid for Specially promoted Research from 2001. 1)Structure of water channel, AQP4, was analyzed by over expression technique of Sf9 cells as well as electron crystallography. Based on the structure we could elucidate mechanisms of orthogonal array formation and cell adhesion of the water channel molecules. 2)Structure and gating mechanism of the acetylcholine receptor pore were analyzed by cryo-electron microscopy (J.Mol.Biol., 319, 1165-1176 (2002), Nature, 423, 949-955 (2003)). 3)A new computer program for automatic particle pickup method of single particle analysis, by which structure of voltage-sensitive sodium channel was analyzed (Nature, 409, 1047-1051 (2001)), was developed (J.Struct.Biol., 136, 227-238 (2001)) and enabled us to analyze structure of IP_3 receptor (J.Mol.Biol., 336, 155-164 (2004)). 4)Structure of Homer was analyzed by X-ray crystallography (J.Mol.Biol., 318, 1117-1126 (2002)), and we developed a new Co-localization Expression Technique (Co-LET) for purification of membrane proteins without detergents (BBRC, 295, 756-765 (2002)). 5)We studied interaction of endothelin B receptor with caveoline-1 (Eur.J.Biochem., 270, 1816-1827 (2003)). Structure of rhodopsin was analyzed at 2.6 Å (Proc.Natl.Acad.Sci.USA, 99, 5982-5987 (2002)). The mutations of three residues in connexin26 were studied to obtain the structural insights of the molecule (J.Boil.Chem., 278, 1807-1816 (2003)). The importance of the second PDZ domain for clustering of PSD-95 was revealed (J.Biol.Chem., 277, 3640-3646 (2002)).