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Structural Analysis of Membrane Proteins by UV Raman Linear Intensity Difference Spectroscopy

紫外拉曼线性强度差光谱法对膜蛋白的结构分析

基本信息

批准号：
11440169
负责人：
TAKEUCHI Hideo
金额：
$ 10.05万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

We applied UV Raman linear intensity difference (RLID) spectroscopy to structural analysis of membrane proteins. First, we developed a new UV Raman spectrometer to obtain high-quality spectra from oriented-membrane samples. The spectrometer was used to analyze the structures of two membrane proteins : bacteriorhodopsin (BR), a proton pump, from Halobacterium salinarium and M2 ion channel protein from influenza A virus. Membranes containing BR were oriented on a glass coverslip and examined by RLID spectroscopy. RLID spectra of a mutant of BR, W189F, was also recorded to extract structural information about Trp189, which is located near the proton release channel. The orientation of the indole ring of Trp189 determined by the RLID method was in agreement with that determined by X-ray diffraction in the crystalline state. Paralleling experiments on wild-type BR and the mutant W189F in the photointermediate M state revealed a reorientation of the indole ring upon release of a proton from the protein. The direction of reorientation of the Trp189 indole ring is opposite to that reported by X-ray diffraction, suggesting structural difference between the membrane state and the crystalline state. The present study demonstrates the utility of RLID spectroscopy in detailed analysis of dynamical structures of membrane proteins. The activation mechanism of the M2 ion channel was also examined by UV Raman spectroscopy. The results shows that protonation of a His residue and its concomitant interaction with a Trp residue is the key step in the activation of the channel. A further analysis of the channel structure by the RLID method is in progress.

我们将紫外拉曼线性强度差光谱（RLID）应用于膜蛋白的结构分析。首先，我们开发了一种新的紫外拉曼光谱仪，以获得高质量的光谱取向膜样品。利用该光谱仪分析了盐生盐杆菌（Halobacterium salinarium）的细菌视紫红质（BR）和甲型流感病毒（Influenza A virus）的M2离子通道蛋白的结构。将含有BR的膜在玻璃盖玻片上取向，并通过RLID光谱法进行检查。还记录了BR突变体W189 F的RLID光谱，以提取关于位于质子释放通道附近的Trp 189的结构信息。通过RLID方法测定的Trp 189的吲哚环的取向与通过X-射线衍射在结晶状态下测定的取向一致。野生型BR和突变体W189 F在光中间体M状态下的标记实验揭示了从蛋白质释放质子后吲哚环的重新取向。的Trp 189吲哚环的重新取向的方向是相反的，通过X-射线衍射报告，表明膜状态和结晶状态之间的结构差异。本研究表明，RLID光谱的膜蛋白的动态结构的详细分析的实用程序。通过紫外拉曼光谱研究了M2离子通道的激活机制。结果表明，质子化的His残基及其伴随的相互作用与色氨酸残基的通道激活的关键步骤。正在进一步分析的RLID方法的通道结构。