MEMBRANE BASIS OF VISUAL EXCITATION

描述(由申请人提供)：该项目的科学重点是描述在膜水平上产生由视紫红质触发的视觉信号转导的事件。其意义在于，视紫红质是G蛋白偶联受体(GPCRs)的原型，而G蛋白偶联受体是45%已知药物的靶标。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类会影响视力疾病。在这里，我们将检验视网膜配基的扭转变形涉及到与视蛋白载脂蛋白的特定结合作用的假设。配体的结合腔有三个位置，即。包括2-紫罗兰酮环、多烯链和质子化的席夫碱。光解触发光感受器的激活，而光感受器与弹性膜变形相耦合。该方法将应用一种关键的生物物理技术--氢(2H)核磁共振波谱，来研究含有视紫红质的、具有重氢视网膜生色团的排列膜。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。来自2H核磁共振的角度限制将照亮视紫红质的暗状态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的分子动力学(MD)模拟和2H核磁共振弛豫测量。这些方法将结合在一起，在膜水平上产生视紫红质激活的全面图像。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2H核磁共振技术应用于排列的膜样品，以阐明视紫红质在光激活Meta II状态下的3D结构和迁移率。将确定Meta II中视网膜的构象和方向，并将其与上一个资金周期中获得的暗态进行比较。(2)下一步，固体~2H核磁共振波谱将研究在视紫红质、Lumi、Meta I和Meta II状态下视网膜发色团的构象扭曲。一个新的方面是研究光解途径中扭曲的视网膜配体的渐进性松弛。(3)第三，视紫红质结合腔内稳定激活的Meta II状态的视网膜的非键合相互作用将用显著改变Meta I Meta II平衡的修饰维甲酸来研究。(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导致视紫红质激活的漂白途径的渐进性松弛。配体的非键相互作用将通过弛豫时间测量来研究，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激活是如何由膜脂双层的弹性变形所支配的。在这里，2H和31P核磁共振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，我们打算为视紫红质和双层脂质如何在视网膜盘膜中触发视觉感知提供一个创新的新视角，作为GPCR和一般信号转导的范例。与公共健康相关：视紫红质是G蛋白偶联受体(GPCRs)的原型，它与生物信号有关，构成了所有已知药物的45%的靶标。该项目将采用一种创新的方法，使用固态2H核磁共振光谱来照亮膜水平的视觉功能。人类遗传性失明可以通过对视紫红质的结构研究来理解，将这种方法扩展到其他GPCR可以刺激基于配体的药物发现。此外，对视网膜盘膜中高度多不饱和的I-3脂类的研究具有潜在的深远意义。必需脂肪酸(EFA)缺乏会影响认知技能和学习。在全国范围内，人们非常关注由于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。了解视膜中的蛋白质和脂肪成分对预防人类疾病具有重要意义。

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.