MEMBRANE BASIS OF VISUAL EXCITATION

描述（由申请人提供）：本研究的科学重点是在分子水平上表征导致视紫红质（一种 G 蛋白偶联受体）触发视觉信号转导的事件。从基本角度来看，所获得的新生物物理学知识具有重要意义，对于色素性视网膜炎等视觉疾病也具有重要意义。结构-功能相结合的方法将阐明视觉信号传导的膜基础。视紫红质是一种完整的膜蛋白，是包含多不饱和脂质双层的超分子组装体的一部分，具有特征性的极性头基组成。因此，视紫红质的结构特性以及膜脂成分对于理解视觉信号转导机制都很重要。为了测试上述概念框架，我们将主要采用一种关键的生物物理方法，即氘（2H）核磁共振波谱。首先，我们计划使用固态 2H NMR 技术来研究视紫红质具有氘化视网膜发色团的重组膜。将结合最近发表的X射线晶体结构，研究视紫红质暗适应基态下视网膜的局部构象、取向和迁移率。新型固态核磁共振方法将包括对齐膜的半随机分布的线形模拟以及弛豫测量。接下来，对齐膜样品的固态 2H NMR 光谱将用于研究红视紫红质、Meta I 和 Meta II 状态下的视网膜发色团。一个创新的新方面是阐明视紫红质（视觉信号转导的触发因素）Meta I-Meta II 转变所伴随的变化。第三，分子动力学计算机技术将被开发并应用于研究膜脂-视紫红质相互作用以及视紫红质3-D结构。其他研究将使用多不饱和膜脂的 2H 和 31P NMR 光谱来区分对 Meta I-Meta II 构象转变影响的替代假设。我们的假设是，这种转变涉及脂质双层曲率弹性应力/应变的变化，从而提供蛋白质能量学与非层状形成膜脂质的性质的耦合。最后，多不饱和膜脂的双层和非层状相的 2H NMR 光谱将研究它们与视紫红质功能相关的平衡和动态特性。因此，我们打算全面了解视紫红质与双层脂质如何在脊椎动物视杆中触发视觉兴奋，这是 G 蛋白偶联受体和信号转导的范例。

DESCRIPTION (provided by applicant): The scientific focus of this research is to characterize the events at the molecular level which lead to the triggering of visual signal transduction by rhodopsin, a G protein-coupled receptor. The new biophysical knowledge to be obtained is significant from a fundamental standpoint, and also with regard to visual diseases such as retinitis pigmentosa. A combined structure-function approach will elucidate the membrane basis of visual signalling. Rhodopsin is an integral membrane protein, and is part of a supramolecular assembly comprising a polyunsaturated lipid bilayer, with a characteristic polar head group composition. As a result, the structural properties of rhodopsin as well as the membrane lipid constituents are both important for understanding the mechanism of visual signal transduction. To test the above conceptual framework, we shall mainly employ a key biophysical methodology, deuterium (2H) NMR spectroscopy. First, we plan to use solid state 2H NMR technology to investigate recombinant membranes in which rhodopsin has a deuterated retinal chromophore. The local conformation, orientation, and mobility of retinal will be studied in the dark-adapted ground state of rhodopsin in conjunction with the recently published X-ray crystal structure. Novel solid-state NMR methods will include lineshape simulations of semi-random distributions of aligned membranes, together with relaxation measurements. Next, solid-state 2H NMR spectroscopy of aligned membrane samples will be used to investigate the retinal chromophore in the bathorhodopsin, Meta I, and Meta II states. An innovative new aspect is to elucidate the changes that accompany the Meta I-Meta II transition of rhodopsin, the trigger for visual signal transduction. Third, molecular dynamics computer technology will be developed and applied to investigate membrane lipid-rhodopsin interactions in conjunction with the rhodopsin 3-D structure. Additional research will use 2H and 31P NMR spectroscopy of the polyunsaturated membrane lipids to distinguish alternative hypotheses for their influences on the Meta I-Meta II conformational transition. Our hypothesis is that the transition involves a change in the curvature elastic stress/strain of the lipid bilayer, thus providing for a coupling of the protein energetics to the properties of non-lamellar forming membrane lipids. Lastly, 2H NMR spectroscopy of bilayers and non-lamellar phases of polyunsaturated membrane lipids will investigate their equilibrium and dynamical properties in relation to rhodopsin function. Thus we intend to provide a comprehensive picture of how rhodopsin together with the bilayer lipids yields triggering of visual excitation in the vertebrate rod, which is a paradigm for G protein-coupled receptors and signal transduction in general.