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

为了在原子和/或亚分子水平上阐明受体G蛋白偶联的分子机制，我们利用光谱、生物化学和分子生物学技术研究了视色素的结构及其与G蛋白的相互作用，得到了以下结果.视色素与G蛋白复合物形成过程的研究。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，但不诱导G蛋白的GDP-GTP交换反应，而交换反应发生在晚期的中间体。振动光谱揭示了视紫红质与G蛋白的相互作用位点以及相互作用位点的结构约束。使用突变的视紫红质，视紫红质的蛋白质区域是G蛋白激活和G蛋白选择性的关键。 ...更多信息 分别鉴定了燕麦蛋白亚型.视色素及其类似物的结构分析。对纯化条件和结晶条件进行了研究，得到了适合于X射线衍射分析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结合冷冻电镜技术，获得了细菌视紫红质的三维结构。在大肠杆菌中大量表达了细菌趋光性受体蛋白pharaonis phoborhodopsin，并鉴定了负责光谱调谐和热行为的氨基酸残基.新型感光蛋白的鉴定及信号转导级联反应调控蛋白的功能分析。利用cDNA克隆技术，新鉴定了存在于脑深部（下丘脑）的光感受蛋白。本文对鸡松果体中的光感受蛋白--视松素的功能进行了研究，并与视网膜中的视色素进行了比较。此外，S-调节蛋白激酶，磷酸二酯酶的特点。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