X-ray Crystallographic Analyses of Retinal Proteins

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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.