X-ray Crystallographic Analyses of Retinal Proteins

细菌视紫红质是在盐生盐杆菌中发现的一种跨膜蛋白，具有光驱动质子泵的功能。它由7个跨膜螺旋组成，含有一个视黄醇分子。这种蛋白质已被广泛研究的各种生物物理技术，其结构和功能已被详细阐明。反应中间体的结构分析对于更好地理解质子泵机制是必不可少的，并且期望以高分辨率确定它们的结构。我们的小组已经开发了一种新的结晶技术，所谓的膜融合方法，通过该方法可以制备含有天然脂质的细菌视紫红质的三维晶体。通过减少X射线损伤的可能影响，我们成功地将结构测定的分辨率提高到可以可视化蛋白质内的水分子的水平。在本项目中，我们对细菌视紫红质的L中间体进行了晶体学分析。发现该中间体的形成伴随着以下结构变化：1）氨基酸残基（Leu 93和Trp 182）与视黄醛的C13甲基接触时发生显著的结构变化（其侧链的旋转和/或置换）; 2）最初与视黄醛席夫碱相互作用的水分子移动到由Leu 93侧链旋转产生的微腔中。此外，我们进行了X-射线晶体学分析的古视紫红质，类似于细菌视紫红质的蛋白质。与细菌视紫红质相比，古菌视紫红质的质子释放通道更加开放。此外，我们还对鱿鱼视紫红质进行了结晶，并收集了低分辨衍射数据。

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.