Analysis of Receptor G-protein coupling in the signal Transduction System of Atomic Resolution.

为了在原子和/或亚分子分辨率上阐明受体G蛋白偶联的分子机制，我们利用多种光谱、生化和分子生物学技术对视觉色素的结构及其与G蛋白的相互作用进行了研究，得到了以下结果。视觉色素与G蛋白形成复合体过程的研究。从低温光谱中，我们确定了视紫红质与G蛋白以不同的方式相互作用的两个中间态。早期的中间体可以与G蛋白结合，但不会引起G蛋白中的GDP-GTP交换反应，而在后期的中间体发生交换反应。振动光谱揭示了视紫红质与G蛋白的相互作用部位以及相互作用部位的结构约束。利用突变的视紫红质，视紫红质的蛋白质区域对于G-蛋白激活和G-Pr…的选择性是必不可少的分别鉴定出更多的Otein亚型。视觉色素及类似物的结构分析。对提纯和结晶条件进行了考察，得到了用于X射线结晶学研究的视紫红质三维晶体，并得到了晶体的衍射图。利用细菌视紫红质的二维晶体与电子冷冻显微镜相结合，得到了细菌视紫红质的三维结构。在大肠杆菌中大规模表达了细菌趋光性受体蛋白--法老鱼视紫红质，并鉴定了与光谱调谐和热行为有关的氨基酸残基。新的光感受器蛋白的鉴定和信号转导级联中调节蛋白的功能分析。利用基因克隆的方法，对存在于脑深部(下丘脑)的感光蛋白进行了鉴定。对鸡松果体中的感光蛋白Pinopsin进行了功能分析，并与视网膜中的视色素进行了比较。此外，还对调节蛋白--S-调制素和磷酸二酯酶进行了鉴定。较少

To elucidate the molecular mechanism of receptor G-protein coupling at atomic and/or submolecular resolution, we have investigated the structures of visual pigments and their interaction with G-proteins by means of various spectroscopic, biochemical and molecular biological techniques and the following results were obtained.1. Studies on the process of complex formation between visual pigments and G-proteins. From the low-temperature spectroscopy, we have identified rhodopsin two intermediate states that interacted with G-protein with different manners. The early intermediate can bind to G-protein but induces no GDP-GTP exchange reaction in G-protein, while the exchange reaction occurs at the later intermediate. The vibrational spectroscopy revealed the interaction site of rhodopsin with G-protein and the structural constraint of the interaction site. Using mutational rhodopsins, the protein region of rhodopsin that are essential for G-protein activation and for the selectivity of G-pr … More otein subtype, respectively, were identified.2. Structural analysis of visual pigments and analogs. The purification and crystallization conditions were examined to get a rhodopsin 3-dimensional crystal for X-ray crystallography and the diffraction pattern of the crystal was obtained. Using 2-dimensional crystal of bacteriorhodopsin in combination with electron cryo-microscopy, the 3-dimensional structure of bacteriorhodopsin was obtained. The large scale expression of pharaonis phoborhodopsin, the receptor protein for phototaxis in bacteria, were performed in E-coli and amino acid residues responsible for the spectral tuning and thermal behavior were identified.3. Identification of the novel photoreceptor proteins and functional analysis of the regulatory proteins in the signal transduction cascade. The photoreceptive proteins present in deep brain (hypothalamus) were newly identified by mean of cDNA cloning. The functional analysis of pinopsin, the photoreceptive protein in chicken pineal, was performed in comparison with those the visual pigments in retinas. Also, S-modulin, the regulator protein to kinase, and phosphodiesterase were characterized. Less