STEREOCHEMISTRY OF VISUAL TRANSDUCTION

细胞如何对细胞外信号作出反应， 细胞内反应在每一个细胞的生命周期中起着关键作用。 cell.将异源三聚体G蛋白偶联至 上游七螺旋跨膜受体和下游效应物 代表了这类细胞信号级联的主要类别。在 根据建议，牛视网膜视杆细胞的视觉级联将 作为这种G耦合信号级联的工作模型。的 该提案的重点是定义伴随的立体化学事件 光信号从视紫红质到异源三聚体G- 蛋白转导素在这项建议中，将处理两个主要领域， 结合晶体学和动力学实验方法。的 控制α-Gt-alpha转导蛋白的立体化学原理 机制将阐明：（a）分析抑制的结构 在通过酶促法使Arg 174不可逆ADP-核糖基化后的GT-α、GTP-γ-S 霍乱毒素，（B）使用可光活化的“笼状GTP”进行 构象变化的时间分辨晶体学分析 伴随GTP的水解，以及（c）分析 GT-α GDP和钒酸盐之间的过渡态类似物复合物。的 GT-α与视紫红质的相互作用将通过以下方式解决：（a） 确定伴随GT-α GDP形成的动力学常数 和GT-α. GTP-γ-S与模拟视紫红质的肽的复合物 相互作用的环和（B）确定Gt-α GDP的结构和 GT-α. GTP-γ-S与模拟视紫红质的肽的复合物 相互作用的循环。 拟议工作的目标是提供一个结构框架， 了解G-耦合信号级联的行为， 特别强调了GTP水解的机制和功能 异源三聚体G蛋白与七螺旋跨膜蛋白的偶联 受体。这种受体在人体生理学中的重要作用 疾病将受益于我们结合动力学和结构 approach.这项工作还可能提出一种解决以下问题的方法： 视网膜色素变性，往往与突变， 感光视紫红质

How a cell responds to an extra-cellular signal and mounts an intracellular response plays a pivotal role in the life cycle of every cell. Signal transduction pathways coupling heterotrimeric G-proteins to upstream seven-helix transmembrane receptors and downstream effectors represent a major class of such cellular signaling cascades. In the following proposal, the visual cascade of the bovine retinal rod cell will serve as the working model for such G coupled signaling cascades. The focus of this proposal is to define the stereochemical events accompanying the passage of a light signal from rhodopsin to the heterotrimeric G- protein transducin. In this proposal two major areas will be addressed by combined crystallographic and kinetic experimental approaches. The stereochemical principles governing transducin alpha's (Gt-alpha) GTPase mechanism will be elucidated by: (a) analyzing the structure of inhibited Gt-alpha.GTP-gamma-S after irreversible ADP-ribosylation of Argl74 by cholera toxin, (b) employing photoactivatable "caged GTP" to carry out a time resolved crystallographic analysis of the conformational changes accompanying hydrolysis of GTP, and (c) analyzing the structure of a transition state analog complex between Gt-alpha.GDP and vanadate. The interactions of Gt-alpha with rhodopsin will be addressed by: (a) determining the kinetic constants accompanying formation of Gt-alpha.GDP and Gt-alpha.GTP-gamma-S complexes with peptides mimicking rhodopsin's interacting loops and (b) determining the structure of Gt-alpha.GDP and Gt-alpha.GTP-gamma-S complexes with peptides mimicking rhodopsin's interacting loops. The goal of the proposed work is to provide a structural framework for understanding the behavior of G-coupled signaling cascades with a particular emphasis on the mechanism of GTP hydrolysis and the functional coupling between heterotrimeric G-proteins and seven helical transmembrane receptors. The central involvement of such receptors in human physiology and disease will benefit from our combined kinetic and structural approach. The work may also suggest a means of tackling the problem of retinitis pigmentosa that is often correlated with mutations in the photoreceptor rhodopsin.