Role of Water Molecules in Proton Transfer in Bacteriorhodopsin

本研究旨在揭示细菌视紫红质（bR）中水的结构变化与其质子泵功能之间的关系。突变体或同位素标记的bR的傅里叶变换红外（FTIR）光谱提供了以下结果。(1)我们建立了红外测量系统，以获得bR及其中间体之间的“无噪声”差谱。此外，时间分辨傅立叶变换红外光谱，偏振傅立叶变换红外光谱，和检测蛋白质-蛋白质相互作用的傅立叶变换红外光谱现在可能应用于视网膜蛋白质，提供更多的信息，其结构的变化。(2)结合bR突变体和FTIR研究了水分子在bR中的位置，确定了沿着质子通道存在的四个水分子的位置。(3)其O-H伸缩振动位于3643 cm-1处的水分子<-1>在形成L-中间体时改变H-键。我们发现，水的结构变化是必不可少的质子转移从视网膜席夫碱Asp 85。(4)我们确定Glu 204是质子释放基团。此外，FTIR光谱揭示了Asp 85和Glu 204之间的氢键网络，在该区域包括水分子。(5)我们发现，单个氨基酸取代将质子泵bR转化为氯离子泵，如盐视紫红质（hR）。很可能bR和hR共享离子泵送的共同机制，并且仅单个氨基酸决定泵送性质。

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.