Conformational Changes Leading to Rhodopsin Activation

描述(申请人提供)：视紫红质，在昏暗的光脊椎动物的光感受器中，是已知最大的细胞表面受体家族中最典型和研究最深入的成员，G蛋白偶联受体(GPCRs)。像视紫红质一样，这些受体都含有一个七螺旋跨膜(TM)结构域、一个细胞质结构域和一个细胞外(视紫红质)结构域，在这个结构域中所有的蛋白质-蛋白质相互作用都发生在信号转导中。最近，视紫红质的暗态结构已经在原子分辨率下得到了解决。这使得人们更加专注于了解视紫红质从黑暗到激活状态发生的精确结构变化。这对于从分子上理解视紫红质的视觉转导和了解激活gpr的相应机制是很重要的。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时TM和细胞质结构域的构象变化。提出了三种实验方法。首先，我们将制备细胞质和TM结构域中每一种氨基酸的半胱氨酸取代突变体，并比较它们在黑暗中和光照后对巯基试剂的反应性。这种半胱氨酸可及性变化的全面图谱将定义黑暗状态和激活状态之间的构象变化。在第二种方法中，可以结合11-顺式和全反式视网膜并且在黑暗中活跃的视紫红质的结构性活性突变体将被研究其构象与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其三级结构中的接近程度。第三种方法将涉及视紫红质的三个突变体的结晶，该突变体结合了三个结构性突变，稳定地与全反式视网膜结合，并至少在与WT视紫红质相同的水平上显示转导蛋白激活。

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.