Structural Studies in Rhodopsin and G Protein Activation

G蛋白偶联受体是一种普遍保守的细胞外信号转导机制。 信号可以通过质膜进行传递。它们占人类基因组的~3%和~50% 在所有非抗菌治疗药物中，作用于GPCR或其途径。当激动剂结合时，结构 改变激活了gpr，并允许gpr结合并诱导α上的核苷酸交换。 异三聚体G蛋白复合体的亚基。视紫红质可能是最能被理解的 通过配体激活gpr，以及随后结合和诱导核苷酸交换 它的同源G蛋白的Gα亚基，转导蛋白。此外，视紫红质与视紫红质：转导 复合体已被用作原型的gpr来模拟一般的激活过程。 GPCRs。 而视紫红质的基态和几个光中间态的结构已经被 通过各种结构技术来解决，我们的知识中存在着一个显著的差距。为了充分地 我们还需要了解视紫质的激活过程，以确定准确的分子 相互作用导致(1)视紫质从基态(暗)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因素以及 激活。需要Meta II激活状态的高分辨率结构才能完全理解序列 包括视紫红质激活的事件。同样，大量的生化和生物物理研究 关于转导蛋白和G蛋白的激活已经进行了，但没有这个复合体的结构，它是 很难理解结合和核苷酸交换的潜在分子基础。这项提议旨在 确定视紫红质结构随其向激活状态转变而发生的根本变化 Meta II状态。此外，我们将确定视紫红质和转导蛋白之间的复合体的结构， 这将有助于阐明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.