Structural Studies in Rhodopsin and G Protein Activation

描述（由申请人提供）：G蛋白偶联受体（GPCR）是一种普遍保守的信号传导机制，细胞外信号可通过该机制跨质膜转导。它们占人类基因组的约3%，约50%的非抗菌治疗剂作用于GPCR或其途径。在结合激动剂后，结构变化激活GPCR，并允许GPCR结合并诱导异源三聚体G蛋白复合物的α亚基上的核苷酸交换。视紫红质可能代表了最好理解的GPCR，用于通过配体激活GPCR以及随后结合并诱导其同源G蛋白转导素的G α亚基上的核苷酸交换。此外，视紫红质和视紫红质：转导素复合物已被用作原型GPCR，以模拟所有GPCR中的一般激活过程。 虽然已经通过各种结构技术解决了视紫红质的基态和几种光中间态的结构，但在我们的知识中存在着显著的差距。为了充分理解视紫红质的激活过程，我们需要确定负责（1）视紫红质从基态（暗态）到完全激活（Meta II）状态的过程以及（2）转导蛋白结合和核苷酸交换和激活的结构决定因素的精确分子相互作用。需要Meta II激活状态的高分辨率结构以完全理解包括视紫红质激活的事件序列。同样，大量的生物化学和生物物理学研究转导和G蛋白的活化已经进行，但没有这种复合物的结构，很难理解的结合和核苷酸交换的潜在分子基础。该建议旨在确定视紫红质结构的根本变化，伴随其过渡到活化的Meta II状态。此外，我们还将确定视紫红质和转导素之间的复合物的结构，这将阐明G蛋白结合和激活的分子机制。 公共卫生相关性：对允许蛋白质视紫红质感知光的分子相互作用的结构研究将大大增加我们对该系统如何发挥作用以及一些致盲疾病的潜在病理学的了解。此外，由于视紫红质是G蛋白偶联受体超家族的成员，它将有助于解释重要的生理过程，如视觉，嗅觉，味觉;心率和新陈代谢是如何控制的。

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) are a universally conserved signalling mechanism by which extracellular signals can be transduced across the plasma membrane. They make up ~3% of the human genome and ~50% of all non-antimicrobial therapeutics act upon GPCRs or their pathways. Upon binding of agonist, structural changes activate the GPCR, and allow the GPCR to bind and induce nucleotide exchange upon the alpha subunit of the heterotrimeric G protein complex. Rhodopsin represents perhaps the best understood GPCR for both GPCR activation by ligand and for the consequent binding and induction of nucleotide exchange upon the G alpha subunit of its cognate G protein, transducin. Furthermore, rhodopsin and the rhodopsin: transducin complex has been used as a prototypical GPCR to model the process of activation in general among all GPCRs. While structures of both the ground state and several photo intermediate states of rhodopsin have been solved through a variety of structural techniques, a significant gap exists in our knowledge. In order to fully understand the process of activation in rhodopsin as well we need to determine the precise molecular interactions that are responsible for (1) the process of rhodopsin going from the ground (dark) state to the fully active (Meta II) state and (2) the structural determinants for transducin binding and nucleotide exchange and activation. A high resolution structure of the Meta II activated state is needed to fully understand the sequence of events that comprises rhodopsin activation. Similarly, a large volume of biochemical and biophysical studies on transducin and G protein activation have been conducted, but without a structure of this complex, it is difficult to understand the underlying molecular basis for binding and nucleotide exchange. This proposal seeks to determine the fundamental changes in rhodopsin structure that accompany its transition to the activated Meta II state. Furthermore, we will determine the structure of the complex between rhodopsin and transducin, which will elucidate the molecular mechanisms of G protein binding and activation. PUBLIC HEALTH RELEVANCE: Structural studies of the molecular interactions that allow the sensing of light by the protein, rhodopsin, will greatly increase our knowledge of how this system functions as well as the underlying pathologies of some blinding diseases. Furthermore, as rhodopsin is a member of the G protein coupled receptor superfamily, it will serve to explain how important physiological processes, such as sight, smell, taste; heart rate and metabolism are governed.