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里叶变换红外（FTIR）光谱。京都、美国和以色列的小组讨论了实验计划。所得结果总结如下。(1)在形成细菌视紫红质的M中间体时，从Glu 204释放质子。FTIR光谱显示Asp 85和Glu 204之间存在氢键网络。新发现一个水分子在Arg 82-Glu 204区域起作用。(2)应用FTIR光谱检测胞质侧Val 49-Thr 46-Asp 96区域的氢键网络。通过使用突变体和同位素标记的蛋白质，揭示了水分子和肽羰基在L中间体的结构变化中的作用。(3)FTIR光谱揭示了阴离子泵菌视紫红质突变体D85 T的活性中心的水分子与氯离子的复合物。(4)在视紫红质的光激发下，一种称为后视紫红质-II的中间体通过特异性蛋白质-蛋白质相互作用激活转导蛋白。我们发明了一个系统来观察transducin和metarhodopsin-II之间的复合物的光谱。检测到肽骨架的结构变化。

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.