Structural Basis of Rhodopsin Function

描述（由申请人提供）：视紫红质是负责昏暗视觉的光感受器，代表了G蛋白偶联受体（gpcr）大超家族的范例。视紫红质的突变与各种视网膜营养不良有关，而引起视紫红质的组成活性的突变与先天性静止性夜盲症有关，这一事实证明了视紫红质的重要性。视紫红质具有典型的7个跨膜螺旋，它们描绘了11顺式视网膜发色团的口袋，该发色团通过质子化的希夫碱与Lys296结合。基态牛视紫红质的x射线结构已被阐明到2.8A分辨率。确定这种高分辨率x射线结构是了解视紫红质功能的重要一步；然而，阐明其激活机制需要了解沿反应途径的中间体的高分辨率结构，以及它们与转导蛋白相互作用的模式。具体来说，我们将询问视网膜光异构化如何触发导致转导蛋白激活的蛋白质结构变化，从而启动视觉感觉。我们的主要假设是，光触发了视网膜的快速异构化，蛋白质既能适应这种变化，又能随后放大成信号传递所需的更大的结构变化。我们处于一个独特的位置来解决这个假设，因为我们最近获得了一个更高分辨率的视紫红质结构，并首次明确地确定了内部水分子。此外，我们在晶体中捕获了紫红质的光中间体，并证明了它们的衍射。该项目的目的是通过解决光中间体深紫红质、光紫红质和后紫红质（后者是视紫红质的信号状态）的x射线结构，以前所未有的空间和时间分辨率绘制出在视紫红质的激活时发生的结构转变。最重要的是，我们将阐明后视紫红质-转导蛋白复合物的晶体结构，以确定该信号单元界面上的相互作用。完成这些具体目标将确定视紫红质活化的物理和化学基础。此外，高分辨率的结构研究将为了解杆状和锥状视觉色素独特的光漂白特性的分子基础开辟道路。

DESCRIPTION (provided by applicant): Rhodopsin is the photoreceptor responsible for dim light vision, and represents the paradigm for the large superfamily of G protein-coupled receptors (GPCRs). The importance of rhodopsin in vision and in medicine is demonstrated by the fact that its mutants are linked to various retinal dystrophies, and mutations causing constitutive activity of rhodopsins are linked to congenital stationary night blindness. Rhodopsin has the canonical seven transmembrane helices, which delineate the pocket for the 11-cis retinal chromophore that is bound to Lys296 via a protonated Schiff base. The X-ray structure of ground-state bovine rhodopsin has been elucidated to 2.8A resolution. Determining this high-resolution X-ray structure was a major step toward developing an understanding of rhodopsin's function; however, elucidating its mechanism of activation requires knowing the high-resolution structures of intermediates along the reaction pathway, as well as their modes of interaction with transducin. Specifically, we will ask how does retinal photoisomerization trigger protein structural changes that lead to activation of transducin, thereby initiating the sensation of vision. Our major hypothesis is that light triggers the rapid isomerization of the retinal, a change which the protein both accommodates and subsequently amplifies into the larger structural changes required for signaling. We are in a unique position to address this hypothesis, as we have recently achieved a higher resolution structure of rhodopsin and have for the first time identified internal water molecules unambiguously. Furthermore, we have trapped photo-intermediates of rhodopsins in the crystals and shown that they diffract. The aims of this project are to map, at unprecedented spatial and temporal resolution, the structural transformations that occur upon activation of rhodospin by solving the X-ray structures of the photo-intermediates bathorhodopsin, lumirhodopsin and metarhodopsin (The latter is the signaling state of rhodopsin). Most importantly, we will elucidate the crystal structure of the metarhodopsin-transducin complex in order to identify the interactions at the interface of this signaling unit. Accomplishing these Specific Aims will define the physical and chemical basis for the activation of rhodopsin. Furthermore, structural studies at high resolution will open the way for understanding the molecular basis for the unique photobleaching properties of rod and cone visual pigments.