Structural Studies in Rhodopsin and G Protein Activation

G蛋白偶联受体（GPCR）是一种普遍保守的信号传导机制，通过它，细胞外 信号可以跨质膜传递。它们占人类基因组的3%，占人类基因组的50%。 的所有非抗菌治疗作用于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α-核苷酸交换。 异源三聚体G蛋白复合物的亚基。视紫红质可能是最好理解的GPCR， 通过配体的GPCR活化以及随后的结合和诱导核苷酸交换， 其同源G蛋白的G α亚单位，转导素。此外，视紫红质和视紫红质： 复合物已被用作原型GPCR，以模拟活化过程，一般在所有 GPCR。 虽然已经研究了视紫红质的基态和几种光中间态的结构， 通过各种结构技术来解决，我们的知识中存在着重大差距。为了充分 了解视紫红质的激活过程，我们需要确定精确的分子 相互作用，负责（1）视紫红质从基态（暗）状态到完全 活性（Meta II）状态和（2）转导蛋白结合和核苷酸交换的结构决定簇， activation.需要Meta II激活态的高分辨率结构来完全理解该序列 包括视紫红质激活。同样，大量的生物化学和生物物理研究 对转导素和G蛋白活化的研究已经进行，但如果没有这种复合物的结构， 很难理解结合和核苷酸交换的潜在分子基础。该提案寻求 以确定视紫红质结构的基本变化，伴随着它的过渡到激活的 Meta II状态。此外，我们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。

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 theraputics 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 photointermediate 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 dificult 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.