STRUCTURAL STUDIES OF VISUAL TRANSDUCTION PROTEINS

视觉转导蛋白的结构研究

• 批准号: 2162121
• 负责人: DANIEL R. KNAPP
• 金额: $ 15.8万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2162121
• 关键词: DNA footprinting; G protein; SDS polyacrylamide gel electrophoresis; conformation; covalent bond; crosslink; cyclic GMP; high performance liquid chromatography; mass spectrometry; membrane proteins; phosphorylation; photoactivation; photochemistry; protein structure function; receptor coupling; rhodopsin; transducin; visual phototransduction

The goal of this work is to understand the visual transduction system at a molecular level in order to define the mechanisms of both normal and abnormal vision. Definition of these mechanisms will provide a basis for design of new therapies for visual diseases. Understanding visual transduction is also of broader biological significance in that the visual receptor rhodopsin is the prototype of the large family of G-protein coupled receptors. This work will employ state of the art mass spectrometry technologies in conjunction with chemical modification and crosslinking experiments to characterize covalent and higher order structures of the receptor protein rhodopsin, its G-protein transducin, its effector enzyme cyclic GMP phosphodiesterase, and the sites of interaction of these proteins in the signal transduction process. There are four Specific Aims: 1. The first aim is to refine methodology for mass spectrometric characterization of covalent modifications on rhodopsin and other membrane proteins. These methods will employ protein cleavage on blotting membranes and on a new type of sample preparation column in conjunction with fragment isolation by both conventional reversed phase HPLC and the newer method of hydrophilic interaction chromatography. 2. The second aim is to probe the three dimensional structure of rhodopsin and the conformational changes which occur upon photoactivation via chemical modification and intramolecular cross-linking experiments. These experiments will employ both photoactivated and heterobifunctional chemical cross-linking reagents to define intramolecular distances, as well as chemical modification experiments to define surface exposed residues, to gain information on the structural changes which occur in rhodopsin upon photoactivation and mutation induced constitutive activation. 3. The third aim is to extend the applicants' previous work on rhodopsin phosphorylation by characterizing all of the sites of phosphorylation at both high and low bleaching levels. 4. The fourth aim is to define the sites of protein-protein interactions in the visual transduction pathway using chemical cross-linking experiments in conjunction with mass spectrometric analysis. These studies will define the sites of interaction between rhodopsin and transducin, and between the transducin alpha subunit and cyclic GMP phosphodiesterase. This work will provide structural information critical to defining the molecular mechanisms of visual transduction, as well as G-protein coupled receptor systems more generally, and will also provide methodology applicable to study of other integral membrane protein systems.

这项工作的目的是了解视觉转导系统 分子水平以定义正常和 异常视力。 这些机制的定义将为 设计新疗法的视觉疾病。了解视觉 转导也具有更广泛的生物学意义，因为视觉 受体视紫红质是G蛋白的大家族的原型 耦合受体。这项工作将采用艺术状态 光谱技术与化学修饰和 交联实验以表征共价和高阶 受体蛋白视蛋白的结构，其g蛋白转丁素， 其效应酶环状GMP磷酸二酯酶，以及 这些蛋白质在信号转导过程中的相互作用。那里 是四个具体目标：1。第一个目的是完善质量的方法 视紫红质和共价修饰的光谱表征和 其他膜蛋白。这些方法将在 在新型样品制备列中涂抹膜和 通过两个常规反向相结合的片段隔离 HPLC和亲水相互作用色谱的新方法。 2。 第二个目的是探测视紫红质的三维结构 以及通过光激活时发生的构象变化 化学修饰和分子内交联实验。 这些 实验将同时采用光活化和异常功能 化学交联试剂以定义分子内距离为 以及化学修饰实验以定义表面暴露 残留物，以获取有关发生的结构变化的信息 光活化和突变诱导的构成型的视紫红质 激活。 3。第三个目的是扩展申请人以前的工作 视紫红质的磷酸化来表征所有的位点 磷酸化在高和低漂白水平上。 4。第四个目标 是在视觉中定义蛋白质 - 蛋白质相互作用的位点 使用化学交联实验的转导途径 与质谱分析的结合。这些研究将定义 视紫红质和跨丁素之间的相互作用位点，以及 跨多霉素亚基和环状GMP磷酸二酯酶。 这项工作将 提供对定义分子至关重要的结构信息 视觉转导的机制以及G蛋白偶联受体 系统更一般，还将提供适用于 其他积分膜蛋白系统的研究。