MEMBRANE BASIS OF VISUAL EXCITATION

描述（由申请人提供）：该项目的科学重点是表征由视紫红质触发视觉信号转导的膜水平事件。重要的是，视紫红质是G蛋白偶联受体（gpcr）的原型，而gpcr是45%的已知药物的靶标。视紫红质突变与黄斑变性和视网膜色素变性有关。此外，视网膜膜的多不饱和脂质影响视力疾病。在这里，我们将测试的假设，扭转变形的视网膜配体涉及特异性结合相互作用与视蛋白载脂蛋白。配体的结合腔具有三个位点，即涉及2-离子环、多烯链和质子化希夫碱。光解触发光感受器的激活，这与弹性膜变形相耦合。该方法将应用一项关键的生物物理技术，氘（2H）核磁共振光谱，来研究含有紫红质和氘化视网膜发色团的排列膜。固态核磁共振方法将包括对膜中蛋白质半随机分布的线形模拟。2H核磁共振的角度约束将照亮视紫质在黑暗和光激活状态下的视网膜结构。进一步的创新需要超大规模分子动力学（MD）模拟和2H核磁共振弛豫测量。这些方法将结合在一起，以产生在膜水平上紫红质活化的综合图像。我们在下一个资助周期的具体目标是：(1)首先，我们计划将固态2H NMR技术应用于排列膜样品，以阐明视紫质在光活化的Meta II状态下视黄醛配体的三维结构和迁移率。Meta II中视网膜的构象和取向将被建立，并与上一个资助周期中获得的暗状态进行比较。(2)接下来，固态2H NMR将研究视网膜发色团在Bathorhodopsin、Lumi、Meta I和Meta II状态下的构象畸变。一个新的方面是研究在光解途径中扭曲的视网膜配体的渐进松弛。(3)第三，通过修饰类维生素a来研究视网膜在视紫质结合腔内稳定活化的Meta II状态的非键相互作用。(4)然后，我们将描述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，并在导致视紫红质激活的漂白途径中逐渐放松。配体的非键相互作用将通过弛豫时间测量进行研究，并通过分子动力学（MD）模拟进一步解释。(5)进一步的研究将阐明视紫红质的光活化是如何由膜脂双分子层的弹性变形所控制的。在这里，2H和31P NMR将研究影响视紫红质光化学功能的膜脂的生物物理性质。因此，我们打算提供一种创新的新观点，即视紫红质如何与双层脂质一起触发视网膜盘膜中的视觉感知，作为gpcr和信号转导的范例。公共卫生相关性：视紫红质是与生物信号有关的G蛋白偶联受体（gpcr）的原型，是所有已知药物中45%的靶标。该项目将采用一种创新的方法，使用固态2H核磁共振光谱来阐明膜水平的视觉功能。人类遗传性失明可以通过对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）缺乏影响认知能力和学习能力。在全国范围内，人们非常关注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的丧失。了解视膜的蛋白质和脂质成分对预防人类疾病至关重要。

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.