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

我们将紫外拉曼线强度差(RLID)光谱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，以获得高质量的取向膜样品的光谱。用该光谱仪分析了盐生盐杆菌的质子泵细菌视紫红质(BR)和甲型流感病毒M2离子通道蛋白两种膜蛋白的结构。将含有BR的膜定向放置在玻璃片上，并用RLID光谱进行检测。还记录了BR突变体W189F的RLID光谱，以提取位于质子释放通道附近的Trp189的结构信息。RLID法测定的Trp189的吲哚环取向与X射线衍射法测定的结晶状态一致。对野生型BR和突变体W189F在光中间M态的平行实验表明，当从蛋白质中释放质子时，吲哚环发生了重新定向。Trp189吲哚环的重取向方向与X-射线衍射法报道的方向相反，表明膜状态和晶态之间存在结构差异。本研究展示了RLID光谱在详细分析膜蛋白的动态结构方面的应用。用紫外拉曼光谱研究了M2离子通道的激活机理。结果表明，His残基的质子化及其与Trp残基的相互作用是通道激活的关键步骤。用RLID方法对通道结构的进一步分析正在进行中。

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.