MEMBRANE BASIS OF VISUAL EXCITATION

描述（由申请人提供）：该项目的科学重点是表征在膜水平上产生视紫红质触发视觉信号转导的事件。重要的是，视紫红质是G蛋白偶联受体（GPCR）的原型，GPCR是45%已知药物的靶点。视紫红质的突变与黄斑变性和视网膜色素变性有关。此外，视网膜盘膜的多不饱和脂质影响视觉疾病。在这里，我们将测试的假设，扭转变形的视网膜配体涉及特定的结合相互作用与视蛋白脱辅基蛋白。配体的结合腔有三个位点，即涉及2-紫罗酮环、多烯链和质子化席夫碱。光分解触发光感受器的激活，这与弹性膜变形相耦合。该方法将应用一个关键的生物物理技术，氘（2 H）NMR光谱，以调查对齐膜含有视紫红质与氘代视网膜生色团。固态NMR方法将包括膜中蛋白质半随机分布的线形模拟。从2 H NMR角度的限制将照亮视网膜结构在黑暗和光激活状态的视紫红质。进一步的创新需要超大规模分子动力学（MD）模拟和2 H NMR弛豫测量。这些方法将结合起来，以产生一个全面的图片视紫红质激活在膜水平。我们在下一个资金周期的具体目标如下：（1）首先，我们计划将固态2 H NMR技术应用于对齐的膜样品，以阐明视紫红质光激活Meta II态中亚视黄醇配体的3D结构和迁移率。将建立Meta II中视网膜的构象和方向，并与上一个资助周期中获得的暗态进行比较。(2)接下来，固态2 H NMR光谱将研究视紫红质、Lumi、Meta I和Meta II状态下视网膜发色团的构象畸变。一个新的方面是研究在光解途径中的扭曲的视网膜配体的渐进松弛。(3)第三，将用显著改变Meta I Meta II平衡的修饰的类维生素A研究视紫红质结合腔内稳定活化的Meta II状态的视黄醇的非键合相互作用。(4)然后，我们将表征视网膜分子动力学的变化，因为它经历了11-顺式到反式异构化和渐进松弛的漂白途径，导致视紫红质的激活。配体的非键相互作用将通过弛豫时间测量进行研究，并进一步解释分子动力学（MD）模拟。(5)进一步的研究将阐明视紫红质的光激活是如何由膜脂双层的弹性变形。在这里，2 H和31 P NMR将研究影响视紫红质光化学功能的膜脂质的生物物理性质。因此，我们打算提供一个创新的视紫红质如何与双层脂质触发视知觉的视网膜盘膜作为一个范例GPCR和信号转导一般。 公共卫生相关性：视紫红质是G蛋白偶联受体（GPCR）的原型，其涉及生物信号传导，并且构成所有已知药物的45%的靶点。该项目将采用一种创新的方法，使用固态2 H NMR光谱来阐明膜水平的视觉功能。人类遗传性失明可以通过视紫红质的结构研究来理解，并且将该方法扩展到其他GPCR可以刺激基于配体的药物发现。此外，对视网膜盘膜高度多不饱和I-3脂质的研究可能具有深远的意义。必需脂肪酸（EFA）缺乏会影响认知技能和学习。在全国范围内，由于人类婴儿饮食中缺乏多不饱和脂肪酸而导致的智力潜力丧失受到极大关注。了解视膜的蛋白质和脂质成分对人类疾病的预防具有重要意义。

DESCRIPTION (provided by applicant): The scientific focus of the project is to characterize the events at the membrane level that yield the triggering of visual signal transduction by rhodopsin. The significance is that rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are the targets of 45% of known pharmaceuticals. Mutations of rhodopsin are implicated in macular degeneration and retinitis pigmentosa. In addition polyunsaturated lipids of the retinal disk membranes affect visual diseases. Here we shall test the hypothesis that torsional deformation of the retinal ligand involves specific binding interactions with the opsin apoprotein. The binding cavity for the ligand has three sites, viz. involving the 2-ionone ring, the polyene chain, and the protonated Schiff base. Photolysis triggers activation of the photoreceptor, which is coupled to elastic membrane deformation. The approach will apply a key biophysical technology, deuterium (2H) NMR spectroscopy, to investigate aligned membranes containing rhodopsin with a deuterated retinal chromophore. Solid-state NMR methods will include lineshape simulations for a semi-random distribution of proteins in the membrane. Angular restraints from 2H NMR will illuminate the retinal structure in the dark and photoactivated states of rhodopsin. Further innovation entails ultra-large scale molecular dynamics (MD) simulations and 2H NMR relaxation measurements. These methods will be combined to yield a comprehensive picture of rhodopsin activation at the membrane level. Our specific aims during the next funding cycle are the following: (1) First, we plan to apply solid-state 2H NMR technology to aligned membrane samples to illuminate the 3D structure and mobility of the retinylidene ligand in the light-activated Meta II state of rhodopsin. The conformation and orientation of retinal in Meta II will be established and compared to the dark-state as obtained in the previous funding cycle. (2) Next, solid-state 2H NMR spectroscopy will investigate conformational distortion of the retinal chromophore in the Bathorhodopsin, Lumi, Meta I, and Meta II states. A new aspect is to investigate progressive relaxation of the distorted retinal ligand in the photolysis pathway. (3) Third, the non-bonded interactions of retinal within the rhodopsin binding cavity that stabilize the activated Meta II state will be investigated with modified retinoids that dramatically shift the Meta I Meta II equilibrium. (4) We will then characterize changes in the retinal molecular dynamics as it undergoes its 11-cis to trans isomerization and progressive relaxation in the bleaching pathway leading to activation of rhodopsin. Non-bonded interactions of the ligand will be investigated through relaxation time measurements and further interpreted by molecular dynamics (MD) simulations. (5) Additional research will illuminate how light activation of rhodopsin is governed by elastic deformation of the membrane lipid bilayer. Here 2H and 31P NMR will investigate the biophysical properties of membrane lipids that influence the photochemical function of rhodopsin. Thus we intend to provide an innovative new view of how rhodopsin together with the bilayer lipids triggers visual perception in the retinal disk membranes as a paradigm for GPCRs and signal transduction in general. PUBLIC HEALTH RELEVANCE: Rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in biological signaling and constitute the targets of 45% of all known pharmaceuticals. The project will adopt an innovative approach using solid-state 2H NMR spectroscopy to illuminate visual function at the membrane level. Human hereditary blindness is can be understood through structural studies of rhodopsin, and extension of the approach to other GPCRs can stimulate ligand-based drug discovery. Moreover, investigations of the highly polyunsaturated I-3 lipids of retinal disk membranes are potentially far-reaching in significance. Essential fatty acid (EFA) deficiency affects cognitive skills and learning. Nationwide there is great concern about the loss of intellectual potential due to lack of polyunsaturated fatty acids in the diets of human infants. Understanding the protein and lipid components of visual membranes is vitally significant to the prevention of human diseases.