STRUCTURAL STUDIES OF VISUAL TRANSDUCTION PROTEIN

视觉转导蛋白的结构研究

• 批准号: 6042684
• 负责人: DANIEL R. KNAPP
• 金额: $ 24.34万
• 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 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6042684
• 关键词: DNA footprinting; G protein; conformation; crosslink; cyclic GMP; electrospray ionization mass spectrometry; high performance liquid chromatography; membrane proteins; membrane reconstitution /synthesis; phospholipids; photoactivation; photochemistry; protein protein interaction; protein structure function; receptor binding; receptor coupling; rhodopsin; transducin; visual phototransduction

DESCRIPTION: (Verbatim from the Applicant's Abstract) The goal of this work is to understand the visual-transduction system at a molecular level 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 because the visual receptor rhodopsin is the prototype of the large family of G-protein couple receptors. Two specific aims are proposed. The first is to characterize the three-dimensional structure of rhodopsin and define the structural changes that occur upon rhodopsin photoactivation. Because rhodopsin is a membrane protein, it is not readily amenable to direct structural approaches such as x-ray crystallography and NMR spectrometry. Instead, indirect methods such as chemical modification topography, chemical cross linking, and tethered reagent cleavage studies in conjunction with mass spectrometry will be used to gain information on solvent-exposed surfaces and distance geometry. Reconstituted membranes containing phospholipids synthesized with photolabeling groups will be used to study motion in the transmembrane region. The results will contribute to the understanding of the structure of rhodopsin and the conformational changes in its cytoplasmic domain that occur upon receptor activation to enable binding of the G protein transducin. The second aim is to characterize structurally the binding of transducin to the activated receptor. These studies will employ surface modification "footprinting" and cross linking to explore how activation of the receptor enables G-protein binding. This work will provide structural information critical to defining the molecular mechanisms of visual transduction and the action of G-protein-coupled receptor systems and will also result in the development of methodology that can be applied to the study of other membrane protein systems.

描述：（逐字研究申请人的摘要）这项工作的目的是 了解分子水平的视觉变换系统以定义 正常和异常视力的机制。这些机制的定义 将为设计新疗法设计视觉疾病提供基础。 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大家族的原型 G蛋白夫妇受体。提出了两个具体目标。首先是 表征视紫红质的三维结构，并定义 视紫红质光活化发生的结构变化。因为视紫红质 是膜蛋白，不容易直接直接结构 X射线晶体学和NMR光谱等方法。反而， 间接方法，例如化学修饰地形，化学交叉 连接和束缚的试剂裂解研究与质量结合 光谱法将用于获取有关溶剂暴露表面和 距离几何形状。合成的含磷脂的重构膜 光标记组将用于研究跨膜的运动 地区。结果将有助于理解结构 视紫红质及其细胞质结构域的构象变化 受体激活后能够结合G蛋白转霉素。这 第二个目的是从结构上表征转霉素与 活化的受体。这些研究将采用表面修饰 “足迹”和交叉链接以探索受体的激活方式 启用G蛋白结合。这项工作将提供结构信息 对于定义视觉转导的分子机制至关重要 G蛋白偶联受体系统的作用，也将导致 开发可以应用于其他膜研究的方法论 蛋白质系统。