Structure and Function Study of Light-Driven Ion Pumps

本研究旨在揭示以视网膜分子为发色团的光驱动离子泵的结构与功能之间的关系。目标分子是作为光驱动质子泵的细菌视紫红质（bR），作为光驱动氯泵的盐视紫红质（hR）和作为光传感器的视紫红质（Rh）。傅里叶变换红外（FTIR）光谱的这些蛋白质及其突变体和同位素标记分子提供了以下结果。(1)利用FTIR偏振光谱研究了bR的希夫碱和Asp85之间存在的一个水分子的位置和取向，揭示了在质子转移中起重要作用的水通过氢键与蛋白质结合。(2) bR的极化FTIR光谱揭示了光异构化过程中蛋白质侧的结构变化。bR中氢键网络的许多结构特征发生了变化。(3)测定了野生型和突变型蛋白bR中N中间体的水结构变化，发现细胞质区存在功能水。此外，通过极化FTIR光谱还观察到特异性的蛋白质结构变化。(5) bR突变体的时间分辨步进扫描FTIR光谱提供了bR光循环最后阶段出现的中间体0的结构信息。(4)低温FTIR光谱揭示了hR的两个L中间体，它们可能与氯化物泵送中可及性的变化有关。(5)低温FTIR光谱法发现Rh中的水分子。

This research project aims at revealing the correlation between structure and function of light-driven ion pumps that possess retinal molecule as the chromophore. The target molecules are bacteriorhodopsin (bR) as a light-driven proton pump, halorhodopsin (hR) as a light-driven chloride pump, and rhodopsin (Rh) as a light-sensor in our vision. Fourier-transform infrared (FTIR) spectroscopy of these proteins as well as their mutants and isotope-labeled molecules provided the following results.(1) Location and orientation of a water molecule present between the Schiff base and Asp85 of bR were studied by means of polarized FTIR spectroscopy, It was revealed that the water, which plays an important role in proton transfer, is bound to protein through hydrogen bonding.(2) Polarized FTIR spectroscopy of bR revealed the structural changes of protein side upon photoisomerization. Many structural features on hydrogen bonding network in bR are changed.(3) Water structural changes in the N intermediate of bR were measured for the wild-type and mutant proteins, and a functional water at the cytoplasmic region was found. In addition, specific protein structural changes were newly observed by polarized FTIR spectroscopy.(5) Time-resolved step-scan FTIR spectroscopy of bR mutants provided the structural information on the 0 intermediate, an intermediate appearing at the last stage of the photocycle of bR.(4) Two L intermediates of hR were revealed by low-temperature FTIR spectroscopy, which could be correlated with change in accessibility in chloride pumping.(5) Water molecules in Rh were discovered by low-temperature FTIR spectroscopy.