Conformational Changes Leading to Rhodopsin Activation

导致视紫红质激活的构象变化

• 批准号: 6572692
• 负责人: H. GOBIND KHORANA
• 金额: $ 28.96万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6572692
• 关键词: conformation; crosslink; crystallization; cysteine; digitonin; disulfide bond; gene mutation; glycosylation; immunoaffinity chromatography; photoactivation; plasmids; protein protein interaction; protein purification; protein structure; rhodopsin; site directed mutagenesis; transducin; transfection /expression vector; visual photoreceptor; visual phototransduction

DESCRIPTION (provided by applicant): Rhodopsin, in the dim light vertebrate photoreceptor, is the prototypic and best-studied member of the largest known family of cell surface receptors, the G-protein coupled receptors (GPCRs). Like rhodopsin, these receptors all contain a seven helical transmembrane (TM) domain, a cytoplasmic domain, where all protein-protein interactions occur in signal transduction and an extracellular (intradiscal in rhodopsin) domain. Recently, the dark state structure of rhodopsin has been solved at atomic resolution. This sharpens focus on understanding the precise structural changes that occur in rhodopsin from dark to its activated state. This is important for molecular understanding of visual transduction by rhodopsin and for understanding the corresponding mechanisms in activation of GPCR. The aim of all the work herein proposed is to understand the conformational changes in the TM and the cytoplasmic domains that occur from the dark to the activated state of rhodopsin. Three experimental approaches are proposed. In the first, single cysteine substitution mutants of every one of the amino acids in the cytoplasmic and TM domains will be prepared and their reactivities to sulfhydryl reagents will be compared in the dark and after illumination. This comprehensive mapping of the changes in accessibilities of the cysteines will define the conformational changes between the dark and the activated states. In the second approach, constitutively active mutants of rhodopsin that can bind both 11-cis- and all-trans-retinal and are active in the dark will be studied for the changes in their conformations from WT rhodopsin. These studies will involve both cysteine scanning and disulfide crosslinking to deduce proximities between different amino acids in their tertiary structures. The third approach will involve crystallization of a triple mutant of rhodopsin that combines three constitutive mutations, is stably bound to all-trans-retinal and displays transducin activation at least at the same level as WT rhodopsin.

描述（由申请人提供）： 弱光脊椎动物光感受器中的视紫红质是已知最大的细胞表面受体家族 G 蛋白偶联受体 (GPCR) 的原型和研究最充分的成员。与视紫红质一样，这些受体都包含一个七螺旋跨膜 (TM) 结构域、一个细胞质结构域（所有蛋白质-蛋白质相互作用都发生在信号转导中）和一个细胞外结构域（视紫红质中的椎间盘内）结构域。最近，视紫红质的暗态结构已在原子分辨率上得到解决。这使得人们更加关注视紫红质从黑暗状态到激活状态所发生的精确结构变化。这对于从分子角度理解视紫红质视觉转导以及理解 GPCR 激活的相应机制非常重要。本文提出的所有工作的目的是了解视紫红质从黑暗状态到激活状态发生的 TM 和细胞质结构域的构象变化。提出了三种实验方法。首先，将制备细胞质和TM结构域中每个氨基酸的单个半胱氨酸取代突变体，并在黑暗中和光照后比较它们对巯基试剂的反应性。这种半胱氨酸可及性变化的全面绘图将定义暗状态和激活状态之间的构象变化。在第二种方法中，将研究可以结合11-顺式和全反式视黄醛并且在黑暗中具有活性的视紫红质组成型活性突变体，以了解其与WT视紫红质的构象变化。这些研究将涉及半胱氨酸扫描和二硫键交联，以推断不同氨基酸在三级结构中的相似性。第三种方法将涉及视紫红质三重突变体的结晶，该三重突变体结合了三个组成型突变，与全反式视网膜稳定结合，并显示出至少与WT视紫红质相同水平的转导蛋白激活。