Role of Water Molecules in Proton Transfer in Bacteriorhodopsin

本研究旨在揭示细菌视紫红质（bR）的水结构变化与其质子泵送功能之间的关系。突变体或同位素标记的bR的傅里叶变换红外光谱（FTIR）提供了以下结果。(1)构建了红外测量系统，获得了bR及其中间体的“无噪声”差谱。此外，时间分辨FTIR、极化FTIR和FTIR检测蛋白质-蛋白质相互作用现在可能应用于视网膜蛋白质，提供更多关于其结构变化的信息。(2)结合bR突变体和FTIR检测bR中水分子的位置。我们已经确定了四个水分子的位置，它们都沿着质子通道存在。(3) O-H伸缩振动位于3643 cm^<-1>的水分子在l -中间体形成时改变了h键。我们发现水结构的改变在质子从视网膜希夫碱转移到Asp85中是必不可少的。(4)我们鉴定出Glu204为质子释放基团。此外，FTIR光谱显示Asp85和Glu204之间存在氢键网络，该区域包含一个水分子。(5)我们发现单氨基酸取代将质子泵送的bR转化为像盐紫红质（hR）一样的氯泵。很可能bR和hR具有共同的离子泵送机制，只有一个氨基酸决定其泵送性质。

This research project aims at revealing the correlation between water structural changes in bacteriorhodopsin (bR) and its function, proton pumping. Fourier-transform infrared (FTIR) spectroscopy of mutant or isotope-labeled bR provided the following results.(1) We constructed the IR measuring system to obtain "noise-free" difference spectra between bR and its intermediates. In addition, time-resolved FTIR,polarized FTIR,and detection of protein-protein interaction by FTIR are now possibly applied to retinal proteins, providing much more information on their structural changes.(2) Location of water molecules in bR were examined by combination of bR mutants and FTIR.We have assigned the location of four water molecules, which are all present along the proton channel.(3) A water molecule whose O-H stretching vibration is located at 3643 cm^<-1> changes H-bonding upon formation of the L-intermediate. We found that the water structural change is essential in the proton transfer from the retinal Schiff base to Asp85.(4) We indentified Glu204 to be the proton release group. In addition, FTIR spectroscopy revealed hydrogen-bonding network between Asp85 and Glu204, including a water molecule in this region.(5) We found that single amino acid substitution converts proton pumping bR to the chloride pump like halorhodopsin (hR). It is likely that both bR and hR share the common mechanism of ion-pumping and only a single amino acid determines the pump property.