Proton Pumping Mechanism in Bacteriorhodopsin-Analysis by Infrared Spectroscopy and Site-Divected Mutagenesis.

京都小组测量了美国小组制备的细菌视紫红质突变体和光反应后的视紫红质的傅立叶变换红外(FTIR)光谱。京都、美国和以色列三国小组讨论了实验计划。主要研究结果如下：(1)细菌视紫红质的M中间体形成后，Glu204释放出一个质子。FTIR光谱显示Asp85和Glu204之间存在氢键网络。新发现了一个水分子在Arg82-Glu204区域工作。(2)用FTIR光谱研究了胞质侧Val49-Thr46-Asp96区域的氢键网络。利用突变体和同位素标记蛋白质揭示了水分子和多肽羰基在L中间体结构变化中的作用。(3)傅立叶变换红外光谱显示阴离子泵浦细菌视紫红质突变体D85T的活性中心存在水分子与氯离子的络合物。(4)在视紫红质的光激发下，一种名为后紫红质的中间体通过特异的蛋白质-蛋白质相互作用激活转导蛋白。我们发明了一种系统来观察变紫红质-II与转导蛋白之间的络合物的光谱。检测了多肽骨架的结构变化。

The Kyoto Group measured Fourier-transform infrared (FTIR) spectra of bacteriorhodopsin mutants, which were prepared by the American group, and rhodopsin upon photoreactions. The experimental plans were discussed among Kyoto, USA and Israel groups. Results obtained are summarized as follows.(1) Upon formation of the M intermediate of bacteriorhodopsin, a proton is released from Glu204. FTIR spectroscopy revealed hydrogen-bonding network between Asp85 and Glu204. A water molecule is newly found to work in the Arg82-Glu204 region.(2) FTIR spectroscopy was applied to examine hydrogen-bonding network in the Val49-Thr46-Asp96 region of the cytoplasmic side. Role of water molecules and peptide carbonyls in the structural changes in the L intermediate is revealed by use of mutants and isotope-labeled proteins.(3) FTIR spectroscopy revealed a complex of a water molecule with a chloride ion in the active center of an anion-pumping bacteriorhodopsin mutant, D85T.(4) Upon photoexcitation of rhodopsin, an intermediate called metarhodopsin-II activates transducin by specific protein-protein interaction. We invented a system to observe the spectra of the complex between metarhodopsin-II and transducin. The structural changes in the peptide backbone was detected.