X-ray Crystallographic Analyses of Retinal Proteins

细菌视紫红质是一种在盐生细菌中发现的跨膜蛋白，其功能是光驱动的质子泵。它由7个跨膜螺旋组成，含有一个视网膜分子。该蛋白已被各种生物物理技术广泛研究，并对其结构和功能进行了详细的阐述。对反应中间体的结构分析对于更好地理解质子泵浦机制是必不可少的，并且需要以高分辨率确定它们的结构。本课题组开发了一种新的结晶技术，称为膜融合法，利用该技术可以制备含有天然脂类的细菌视紫红质三维晶体。通过减少X辐射损伤的可能影响，我们成功地将结构确定的分辨率提高到可以看到蛋白质中的水分子的水平。在本项目中，我们对细菌视紫红质的中间体L进行了结晶学分析。研究发现，这一中间体的形成伴随着以下结构变化：1)与视网膜C13甲基接触的氨基酸残基(Leu93和Trp182)经历了显著的结构变化(侧链旋转和/或位移)；2)最初与视网膜Schiff碱相互作用的水分子移动到由Leu93侧链旋转产生的微腔中。此外，我们还对一种类似于细菌视紫红质的蛋白质--古紫质进行了X射线结晶学分析。与细菌视紫红质相比，古紫质的质子释放通道更为开放。此外，我们还对鱿鱼视紫红质进行了结晶，并采集了低分辨率的衍射数据。

Bacteriorhodopsin, a transmembrane protein found in halobacterium halobium, functions as a light-driven proton pump. It is composed of 7 transmembrane helices and contains one molecule of retinal. This protein has been extensively studied by various biophysical techniques and its structure and function has been elucidated in detail. Structural analysis of the reaction intermediates is indispensable for better understanding of the proton pumping mechanism and determination of their structures at high resolution has been desired. Our group has developed a novel crystallization technique, so called the membrane fusion method, by which a three-dimensional crystal of bacteriorhodopsin containing native lipids can be prepared. By reducing the possible effect of X-radiation damages, we have succeeded in increasing the resolution of structural determination to such a level that water molecules within the protein can be visualized. In this project, we carried out a crystallographic analysis of the L intermediate of bacteriorhodopsin. It is found that formation of this intermediate accompanies the following structural changes : 1)amino-acid residues (Leu93 and Trp182) contacting the C13 methyl of retinal undergo significant structural changes (rotation and/or displacement of their side chains) ; 2)a water molecule that initially interacts with the retinal Schiff base moves to a micro-cavity that is created by rotation of the side chain of Leu93. Furthermore, we carried out X-ray crystallographic analysis of archaerhodopsin, a protein analogous to bacteriorhodopsin. As compared to bacteriorhodopsin, the proton release channel is more opened in archaerhodopsin. In addition, we crystallized squid rhodopsin and collected a low-resolution diffraction data.