STEREOCHEMISTRY OF VISUAL TRANSDUCTION

细胞如何响应细胞外信号并安装 细胞内反应在每个生物的生命周期中起着举足轻重的作用 手机。异源三聚体G蛋白偶联的信号转导途径 上游七螺旋跨膜受体和下游效应器 代表了这类细胞信号级联的一个主要类别。在 根据提议，牛视网膜视杆细胞的视觉级联将 作为这种G耦合信令级联的工作模型。这个 该提案的重点是定义伴随着的立体化学事件 光信号从视紫红质传递到异三聚体G- 蛋白质转导蛋白。在这项提案中，两个主要领域将由 结晶学和动力学实验相结合的方法。这个 转导蛋白α(GT-α)GTP酶的立体化学原理 其作用机制将通过：(A)分析抑制物的结构 GT-α、GTP-γ-S对Arg74的不可逆ADP-核糖基化反应 霍乱毒素；(B)使用可光激活的“笼养GTP”进行 构象变化的时间分辨结晶学分析 伴随着GTP的水解，以及(C)分析一个 GT-α、GDP和钒酸盐之间的过渡态模拟络合物。这个 GT-α与视紫红质的相互作用将通过以下方式解决：(A) GT-α·GDP形成过程中伴随动力学常数的测定 和GT-α.GTP-γ-S与视紫红质模拟多肽的络合物 相互作用的循环和(B)确定GT-Alpha.GDP和 GT-α、GTP-γ-S与视紫红质模拟多肽的络合物 相互作用的循环。 拟议工作的目标是为 了解G耦合信号级联的行为 重点介绍了GTP的水解机理和功能 异源三聚体G蛋白与七种螺旋跨膜的偶联 感受器。这种受体在人体生理学中的中枢作用 疾病将从我们的动力和结构相结合中受益 接近。这项工作还可能提出一种解决 通常与视网膜色素变性相关的视网膜色素变性 光感受器视紫质。

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.