Structural Analysis of the Rhodopsin-Transducin Complex

视紫红质转导蛋白复合物的结构分析

• 批准号: 7248586
• 负责人: KEVIN Donald RIDGE
• 金额: $ 33.49万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-19 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7248586
• 关键词: Address; Agonist; Binding; Binding Sites; Biological Models; Chimera organism; Complex; Condition; Coupling; Data; Detergents; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Guanine Nucleotides; Helix (Snails); Heteronuclear NMR; Heterotrimeric GTP-Binding Proteins; Isotope Labeling; Lead; Length; Ligands; Maps; Mediating; Methods; Molecular Conformation; Monitor; Mutation; Nuclear Magnetic Resonance; Physiological; Protein Overexpression; Reaction; Research; Resolution; Rhodopsin; Sensory; Signal Transduction; Solutions; Structure; System; Transducin; Visual; Work; base; design; drug discovery; ear helix; milligram; mutant; receptor; receptor binding; reconstitution; research study; seven-transmembrane G-protein-coupled receptor

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are a large group of 7 transmembrane helix receptors that require ligand-dependent activation to initiate heterotrimeric (a, B, y) G-protein mediated intracellular signaling cascades. Due to their physiological relevance and pharmacological tractability, GPCRs are the focus of numerous drug discovery efforts. Activation of a G-protein by its agonist stimulated GPCR (R*) requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide-binding pocket of the G-protein. The structural basis for the interaction of a GPCR with its cognate G-protein, and the subsequent activation of the G-protein by R*, are not fully understood. The overall goal of this research is to develop and apply high-resolution nuclear magnetic resonance (NMR) methods to probe the structural basis for the propagation of structural signals from R* to the G-protein, with a specific focus on elucidating structural changes in the a-subunit that lead to guanine nucleotide exchange. For these studies, signaling of the G-protein, transducin (Gt), by the light-activated GPCR, rhodopsin, will be used as a model system. The interaction of Gt with solubilized native and mutant rhodopsins, as well as soluble mimics of R*, will be employed to allow for the observation and trapping of discrete states accompanying signal transfer. Using isotope-labeled G-protein a-subunits, heteronuclear NMR methods will be used to selectively map structural changes in this subunit upon heterotrimer formation with unlabeled By-subunits and when trapped in discrete R* bound states. 3 fundamental structural questions surrounding the mechanism of R* mediated signal transfer will be addressed: 1. What structural changes in the receptor binding interface of the a-subunit occur upon interaction of the G-protein heterotrimer with R*?; 2. What are the structural changes in the guanine nucleotide binding site that occur upon interaction of the G-protein heterotrimer with R*?; 3. How are structural changes that result from the interaction of the binding interface of the a-subunit with R* correlated to changes in the conformation of the guanine nucleotide binding site? We expect that this work will have a fundamental impact on our understanding of the mechanisms governing activation of G-proteins by R* and form a basis for understanding the structural consequences of naturally occurring rhodopsin and Gt mutations associated with visual dysfunction.

描述(申请人提供)：G蛋白偶联受体(GPCRs)是一个由7个跨膜螺旋受体组成的大群，需要配体依赖的激活来启动异三聚体(a，B，y)G蛋白介导的细胞内信号级联反应。由于其生理相关性和药理上的易操纵性，GPCRs是众多药物发现努力的焦点。G蛋白被其激动剂刺激的GPCR(R*)激活，需要将结构信号从受体结合界面传播到G蛋白的鸟核苷酸结合口袋。GPCR与其同源G蛋白相互作用以及随后由R*激活G蛋白的结构基础尚不完全清楚。这项研究的总体目标是开发和应用高分辨率核磁共振(NMR)方法来探索结构信号从R*传播到G蛋白的结构基础，重点是阐明导致鸟嘌呤核苷酸交换的a亚基的结构变化。在这些研究中，通过光激活的视紫红质G蛋白转导蛋白(GT)的信号将被用作模型系统。GT与增溶的天然和突变视紫红质的相互作用，以及可溶的R*模拟物，将被用来观察和捕获伴随信号传递的离散状态。利用同位素标记的G蛋白a亚基，异核核磁共振方法将被用来选择性地绘制当与未标记的副亚基形成异三聚体时以及当捕获到离散的R*结合态时该亚基的结构变化。围绕R*介导的信号传递机制的3个基本结构问题将被解决：1.当G-蛋白质异三聚体与R*相互作用时，α-亚基的受体结合界面会发生什么结构变化？2.当G-蛋白质异三聚体与R*相互作用时，鸟嘌呤核苷酸结合部位的结构变化是什么？3.由于a-亚基与R*结合界面的相互作用而导致的结构变化与鸟嘌呤核苷酸结合部位构象的变化有何关联？我们预计，这项工作将对我们理解R*激活G蛋白的机制产生根本性的影响，并为理解与视觉功能障碍相关的自然发生的视紫红质和GT突变的结构后果奠定基础。