Elucidation by infrared spectroscopy on the mechanism for the induction of the pumping function in bacteriorhodopsin

细菌视紫红质是一种利用光能进行单向质子运输的蛋白质。我们用傅里叶变换红外光谱分析了这一过程中的结构特征，这对于获得与该功能相关的化学键的信息是有意义的。通过利用定点突变蛋白和同位素替代将它们定位到蛋白质中的特定位置，我们可以揭示其功能表达的机制。在光中间体中，用于质子从席夫碱转移到ASP85的L中间体和用于质子从ASP96转移到希夫碱的M中间体对质子泵浦都是重要的。揭示水分子在这些过程中的作用是我们的突出贡献。我们还揭示了在L到M的转化过程中负责质子释放的残基是面向胞外介质的Glu204。通过使用具有长寿命O中间体的突变蛋白，我们发现蛋白质中的变化可以早于生色团的变化。一种突变蛋白，其中Asp85被Thr取代，获得氯离子泵送活性而不是质子泵送活性。在此基础上，我们揭示了氯在卤视紫质中的转运机制。视紫红质是一种视觉色素，它有一个七螺旋束。细菌视紫红质的类似思想也被用来揭示红景天素活性中心的水分子的结构和作用。还阐明了水分子和多肽羰基参与了变紫红质II与G蛋白的激活过程。

Bacteriorhodopsin is a protein which carries our unidirectional proton transport by use of light energy. We have analyzed structural features in this process by Fourier transform infrared spectroscopy, which is pertinent to obtain the informations on the chemical bonds relevant for the function. By locatiog them to the specific positions in the protein by use of site-specific mutant proteins and isotope substitutions, we can reveal the mechanism for the functional expression. Among the photointermediates, both the L intermediate for the proton transfer from the schiff base to Asp85, and the M intermediate for the proton transfer from Asp96 to the Schiff base are important for the proton pumping. It is our prominent contribution to reveal the involvement of the water molecules in these processes. we also revealed the residue responsible for the proton release in the L-to-M conversion as Glu204, which faces to the extracellular medium. By use of mutant proteins with long-lived O intermediate, we showed that the canges in the protein can occur earlier than the changes in the chromophore. A mutant protein, in which Asp85 is replaced by Thr, acquires chloride pumping activity in place of proton pumping activity. On this basis, we revealed transport mechanism of chloride in halorhodopsin. Visual pigment, rhodopsin, has a seven-helical bundle. Similar idea for bacteriorhodopsin was applied to reveal the structure and role of water molecule in the active center of rhodosin. The involvement of water molecules and peptide carbonyls in the process for the activation metarhodopsin II with the G-protein were also elucidated.