Structural Dynamics of Retinal Binding and Release

描述（由申请人提供）：我们研究的长期目标是了解g蛋白偶联受体（gpcr）被激活和减弱的分子机制。这些受体代表了人类基因组中最大的家族，它们是大多数药物的靶标。我们的研究主要集中在GPCR视紫红质及其附属蛋白上。虽然参与视觉信号的关键蛋白质的晶体结构现在已经知道，但这些蛋白质在其激活和衰减过程中所经历的大多数关键结构变化在很大程度上仍然是一个猜测问题。特别是，我们甚至缺乏有关减弱视紫质信号的动态事件的基本信息，即视网膜从视蛋白结合口袋中释放的机制，以及视网膜的结合和释放如何影响逮捕蛋白的结合和释放。了解这些过程对视觉研究至关重要——视网膜连接的稳定性在不同视蛋白之间差异很大，并且是一些视觉疾病状态的一个因素。此外，尽管人们对抑制蛋白与视紫红质结合的机制和动力学了解甚多，但对抑制蛋白在结合后释放的原因以及这种释放与视网膜发色团状态的关系知之甚少。在本提案的目的1中，我们将确定视紫红质如何控制其视网膜希夫碱键的水解。在Aim II中，我们将研究视网膜摄取和释放如何发生在视紫质中，使用最新的视紫质结构来指导我们的研究。最后，在Aim III中，我们将使用我们的新方法来跟进我们在上一个资助期间的发现-捕获蛋白可以与MIII视紫红质结合，从而捕获和阻止视网膜释放。了解抑制素如何调节视网膜释放对健康至关重要，因为抑制素可能有助于限制在强光条件下游离视网膜的释放，从而有助于限制氧化视网膜加合物的形成，而氧化视网膜加合物可能导致萎缩性年龄相关性黄斑变性（AMD）等疾病。同样地，了解是什么使抑制蛋白在结合视紫红质后“释放”也是至关重要的——稳定的视紫红质-抑制蛋白复合物被认为是细胞凋亡和常染色体显性视网膜色素变性（ADRP）的一个促进因素。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the molecular mechanisms through which G-protein coupled receptors (GPCRs) are activated and attenuated. These receptors represent the largest family in the human genome, and they are the target of most pharmaceutical drugs. We focus our studies primarily on the GPCR rhodopsin and its affiliate proteins. Although crystal structures of key proteins involved in visual signaling are now known, most of the critical structural changes these proteins undergo during their activation and attenuation remain largely a matter of speculation. In particular, we lack even rudimentary information about the dynamic events involved in attenuating rhodopsin signaling, namely, the mechanisms through which retinal is released from the opsin-binding pocket, and how retinal binding and release affects arrestin binding and release. Understanding these processes is of fundamental importance for vision research - the stability of the retinal linkage varies widely among different opsins and is a factor in some visual disease states. Furthermore, although much is known about the mechanism and kinetics of arrestin binding to rhodopsin, little is known about what makes arrestin release after binding, and how this release is related to the status of the retinal chromophore. In Aim I of this proposal we will determine how rhodopsin controls the hydrolysis of its retinal Schiff base linkage. In Aim II we will examine how retinal uptake and release occurs in rhodopsin, using the recent structure of opsin to guide our studies. Finally, in Aim III, we will use our novel methods to follow up on a discovery we made during the last funding period - that arrestin can bind to MIII rhodopsin, thus trapping and preventing retinal release. Understanding how arrestin regulates retinal release is fundamentally important to health, as arrestin may serve to limit the release of free retinal under bright light conditions, and thus help limit the formation of oxidative retinal adducts that can contribute to diseases like atrophic age-related macular degeneration (AMD). Similarly, understanding what makes arrestin "let go" after binding rhodopsin is also crucial - stable rhodopsin-arrestin complexes have been suggested to be a contributing factor in apoptosis and autosomal dominant retinitis pigmentosa (ADRP). PUBLIC HEALTH RELEVANCE: The proposed research will define the molecular events involved in the binding and release of retinal. We will investigate how retinal gets into and out of the binding pocket in rhodopsin, what makes it stay there (i.e., how the Schiff-base linkage forms and hydrolyzes), and how the protein arrestin affects these processes. Answering these fundamental questions will help shed light on more general questions in vision, such as why rates of retinal binding and release vary so greatly between Rod and Cone rhodopsins, and help elucidate underlying mechanisms for several retinal diseases.