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

点击翻译按钮获取中文摘要

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.