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

红外光谱阐明细菌视紫红质泵浦功能诱导机制

基本信息

批准号：
06404082
负责人：
MAEDA Akio
金额：
$ 18.56万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

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.

细菌紫红蛋白是一种蛋白质，它通过使用轻能携带我们的单向质子运输。我们已经通过傅立叶变换红外光谱分析了此过程中的结构特征，这与获得有关该功能相关的化学键的信息是相关的。通过使用位点特异性突变蛋白和同位素取代，我们可以揭示功能表达的机制，通过使用位点特异性突变蛋白和同位素取代，将其置于蛋白质中的特定位置。在光互中的质子中间体中，质子从Schiff碱基到ASP85的L中间体以及从ASP96到Schiff碱基的M中间体对于质子泵送都很重要。这是我们揭示水分子参与这些过程的重要贡献。我们还揭示了负责L-TO-M转化率中质子释放的残留物为GLU204，该残基面向细胞外培养基。通过使用具有长寿命O中间体的突变蛋白，我们表明蛋白质中的罐头可能比发色团的变化早发生。一种突变蛋白，其中ASP85被THR取代，可取代氯化物泵送活性，以代替质子泵送活性。在此基础上，我们揭示了氯哌替丁物中氯化物的运输机制。视觉颜料，视紫红质，有一个七螺旋的束。采用类似细菌淡淡蛋白酶的类似思想来揭示水分子在若多活性中心中的结构和作用。还阐明了水分子和肽羰基在激活元紫红素II与G蛋白的过程中的参与。