MOLECULAR MECHANISMS OF LIGHT TRANSDUCTION BY RHODOPSIN

视紫红质光传导的分子机制

• 批准号: 2404314
• 负责人: Sriram Subramaniam
• 金额: $ 27.65万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-06-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2404314
• 关键词: Drosophilidae; Schiff bases; Sf9 cell line; animal tissue; chemical kinetics; circular dichroism; conformation; crystallization; electron microscopy; gene expression; glycosylation; hydroxylamine; molecular cloning; point mutation; protein folding; protein purification; protein reconstitution; protein structure function; retinaldehyde; retinitis pigmentosa; rhodopsin; site directed mutagenesis; tissue /cell culture; transducin; ultraviolet spectrometry; visual phototransduction

Membrane proteins that function as G-protein coupled receptors play a central role in mediating fundamental processes such as vision, olfaction, taste and touch. The etiology of a number of human diseases have ben traced to mutations that either affect the folding of these proteins into the correct three dimensional structures, or affect their ability to function in signal transduction. While a wealth of biochemical evidence has defined regions of these receptors that are important for ligand binding, G-protein activation, phosphorylation, arrestin binding, and regulation by other signaling proteins, many fundamental questions remain. What is their three-dimensional structure in the membrane? What are the events that occur during their biogenesis which ensure formation of the correct three- dimensional structure? What are the critical protein conformational changes that must occur upon activation? How do disease-causing mutations affect formation of the correct structure and/or steps in signal transduction? The experiments described in this proposal are aimed at answering these questions using rhodopsin, the G-protein coupled light receptor in vertebrates and in invertebrates as a model system. the main goals for this proposal period are to: (I) investigate structural requirements of the correct membrane insertion of newly synthesized bovine rhodopsin by analysis of the topologies of mutant polypeptides with varying lengths, (ii) probe the interaction of the nascent rhodopsin polypeptides with membrane protein components of the endoplasmic reticulum machinery using site-selective photoreactive probes incorporated into the growing polypeptide chain, (iii) initiate efforts to determine the three-dimensional structures of Drosophila Rh1 rhodopsin and metarhodopsin by electron diffraction analysis of two-dimensional structures of Drosophila Rh1 rhodopsin and metarhodopsin by electron diffraction analysis of two-dimensional crystals and (iv) study the mechanism of light-driven activation od Drosophila Rh1 rhodopsin by spectroscopic and biochemical studies of rhodopsin isolated from transgenic flies. I anticipate that together, these experiments will provide molecular "snapshots" of the biogenesis, structure, and mechanisms of activation of rhodopsin.

作为G蛋白偶联受体发挥功能的膜蛋白 在调解基本进程中发挥核心作用， 视觉嗅觉味觉和触觉 一些疾病的病因 人类疾病可以追溯到突变， 将这些蛋白质折叠成正确的三维结构 结构，或影响其在信号中发挥作用的能力 转导 虽然大量的生化证据表明 这些受体的特定区域对于配体的形成是重要的， 结合，G蛋白活化，磷酸化，抑制蛋白结合， 和其他信号蛋白的调节， 问题依然存在。 它们的三维结构是什么 膜？ 在他们的生活中发生了什么 生物起源，确保形成正确的三个- 空间结构？ 关键蛋白质是什么 激活后必然发生的构象变化？ 如何 致病突变是否会影响 结构和/或信号转导步骤？ 本提案中描述的实验旨在 用视紫质来回答这些问题，G蛋白 在脊椎动物和无脊椎动物中， 模型系统。 本提案期的主要目标是： (I)研究正确膜的结构要求 通过分析新合成牛视紫红质的插入 具有不同长度的突变多肽的拓扑结构，（ii） 探测新生视紫红质多肽与 内质网膜蛋白成分 使用位置选择性光反应探针的机械 （iii）启动多肽链的生长， 努力确定的三维结构， 果蝇Rh 1视紫红质和变视紫红质的电子传递 果蝇二维结构的衍射分析 Rh 1视紫红质和变视紫红质的电子衍射 分析二维晶体和（iv）研究 果蝇Rh 1光驱动激活机制 视紫红质的光谱和生物化学研究 从转基因果蝇中分离的视紫红质。 我预计 总之，这些实验将提供分子“快照” 的生物起源，结构和激活机制， 视紫红质