Structure and Function Study of Light-Driven Ion Pumps

本研究旨在揭示以视网膜分子为生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)，作为光驱动氯化物泵的卤视紫红质(HR)，以及作为我们视觉中的光传感器的视紫红质(Rh)。这些蛋白质及其突变体和同位素标记分子的傅里叶变换红外光谱研究结果如下：(1)利用偏振傅里叶变换红外光谱研究了BR的Schiff碱和Asp85之间的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的偏振FTIR光谱揭示了蛋白质光异构化过程中蛋白质侧结构的变化。BR中氢键网络的许多结构特征都发生了变化。(3)测定了野生型和突变型BR中N中间体的水结构变化，并在细胞质区域发现了功能水。此外，偏振傅里叶变换红外光谱还观察到了特定蛋白质结构的变化。(5)BR突变体的时间分辨步进扫描FTIR光谱提供了0中间体的结构信息，该中间体出现在BR光周期的最后阶段。(4)低温傅立叶变换红外光谱揭示了HR的两个L中间体，这可能与氯泵中可及性的变化有关。(5)低温FTIR光谱发现了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.