MEMBRANE BASIS OF VISUAL EXCITATION

视觉兴奋的膜基础

• 批准号: 9006514
• 负责人: Michael F Brown
• 金额: $ 36.48万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9006514
• 关键词: Address; Affect; Affinity; Age-Years; Agonist; Apoproteins; Binding; Binding Proteins; Biochemistry; Biological; Biological Process; Biology; Catalysis; Cellular Membrane; Computer Simulation; Coupled; Coupling; Data; Development; Dimensions; Discrimination; Disease; Elderly; Electronics; Encapsulated; Environment; Equilibrium; Event; Eye diseases; Face; Family; Foundations; Fourier Transform; Functional disorder; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Grant; Health; Human; Human Biology; Hydration status; Hydrogen Bonding; Individual; Investigation; Learning; Length; Ligands; Light; Lipid Bilayers; Lipids; Macular degeneration; Magnetic Resonance; Mechanics; Mediating; Medicine; Membrane; Membrane Lipids; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; Motion; Movement; NMR Spectroscopy; National Institute on Aging; Night Blindness; Odors; Opsin; Organism; Osmotic Pressure; Patients; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Photochemistry; Photons; Physiological; Property; Protein Dynamics; Proteins; Reading; Receptor Activation; Relaxation; Research; Retinal; Retinitis Pigmentosa; Retinoids; Rhodopsin; Roentgen Rays; Role; Shapes; Signal Transduction; Signaling Protein; Site; Societies; Spectrum Analysis; Spin Labels; Stress; Structure; Study models; Surface; Technology; Temperature; Testing; Text; Time; Transducin; Vision; Visual; Visual Acuity; Visual Signal Transduction Pathway; Water; Work; age related; aging population; base; cofactor; cognitive skill; dark matter; drug discovery; flexibility; hereditary blindness; innovation; insight; interest; metarhodopsin; molecular dynamics; molecular size; movie; mutant; novel; novel strategies; peer; peptide analog; polyunsaturated fat; prototype; receptor; receptor function; research study; restraint; retinal rods; simulation; solid state; solid state nuclear magnetic resonance; temporal measurement; visual excitation; visual process; visual processing

DESCRIPTION (provided by applicant): Our investigation aims to establish how the atomic and membrane-level events operate to trigger visual signal transduction by rhodopsin in a unified multi-scale framework, with broad implications for biological signaling. The high impact of understanding G-protein-coupled receptor (GPCR) activation is well appreciated. Yet numerous gaps in our understanding remain, both with rhodopsin, as well as other Family A GPCRs for which rhodopsin is a highly significant prototype. Here we plan to resolve the long sought, critical mechanistic features by an innovative approach that combines magnetic resonance (solid-state 2H and 13C NMR), Fourier transform infrared (FTIR), and electronic (UV-visible) spectroscopy. Our novel hypothesis for rhodopsin activation is formulated in terms of factors that drive a progression of transient conformational substates through an active ensemble: release of retinal strain, retinal-specific protein dynamics, hydration changes, pH catalysis, dynamical G-protein coupling, and membrane stress due to the polyunsaturated lipid composition. (1) Application of our novel solid-state 2H and 13C NMR technology will reveal how the release of conformational strain through retinal isomerization and relaxation unlocks the active rhodopsin (Meta-II) state. The angular and distance restraints from 2H and 13C solid-state NMR will illuminate the dynamical structure of retinal through its progression of active sub-states toward the active Meta-II form. (2) Changes in local retinal dynamics as studied by 2H and 13C NMR relaxation studies will be correlated with rhodopsin's activating motions by combining the results of spectroscopy with molecular dynamics (MD) simulations. Local retinal mobility will be related to large-scale protein dynamics involving fluctuations of the transmembrane helices. Notably, this work will address ambiguous X-ray structural data with results obtained at more physiological temperatures. Our investigation will decide the question of why active Meta-II rhodopsin and the ligand-free Opsin* apoprotein have similar X-ray structures, yet completely different activities. (3) Next we plan to investigate the specific role f pH and hydration throughout the active ensemble in relation to rhodopsin's interaction with transducin. We plan to combine our spectroscopic methods (UV-visible, FTIR, site-directed spin-labeling) with osmotic pressure studies to investigate changes in water-mediated H-bonding networks, together with a dramatic water influx due to transmembrane helical movements in rhodopsin activation. (4) Additional research will uncover the influences of polyunsaturated lipids on rhodopsin through modulation of membrane curvature stress, and how lipid composition biases the distribution of states in the active ensemble mechanism. Our plan encapsulates a new multi-scale view of how rhodopsin initiates visual signal transduction, tying together mechanical, environmental, temporal, and structural factors. Understanding how these factors interoperate across various length and time scales is fundamental to understanding of how rhodopsin achieves the extreme high fidelity required for visual signaling. Our robust and novel approach will provide important insights that are transferable to the broader class of GPCRs in biology and pharmacology.

描述(申请人提供)：我们的研究旨在确定原子和膜水平的事件如何在统一的多尺度框架内触发视紫红质的视觉信号转导，对生物信号具有广泛的影响。了解G蛋白偶联受体(GPCR)激活的高度影响是很好的认识。然而，我们对视紫红质的理解仍然存在许多空白，以及视紫红质是非常重要的原型的其他A家族GPCR。在这里，我们计划通过一种结合了磁共振(固态2H和13C核磁共振)、傅立叶变换红外(FTIR)和电子(UV-可见)光谱的创新方法来解决长期寻求的关键机械特征。我们关于视紫红质激活的新假说是根据通过活性系综驱动瞬时构象亚态进展的因素而提出的：视网膜应变的释放、视网膜特有的蛋白质动力学、水合作用的变化、pH催化、动态G蛋白偶联以及由于多不饱和脂肪组成而导致的膜压力。(1)应用我们的新型固态2H和13C核磁共振技术将揭示通过视网膜异构化和松弛释放构象应变如何解锁活性视紫红质(Meta-II)状态。2H和13C固体核磁共振的角度和距离限制将通过其活性亚态向活性Meta-II形式的进展来阐明视网膜的动态结构。(2)结合光谱学和分子动力学(MD)模拟的结果，通过2H和13C核磁共振弛豫研究局部视网膜动力学的变化将与视紫红质的激活运动相关联。局部视网膜移动性将与涉及跨膜螺旋波动的大范围蛋白质动力学有关。值得注意的是，这项工作将用在更多生理温度下获得的结果来解决模糊的X射线结构数据。我们的研究将决定为什么活性的Meta-II视紫红质和无配体的Opsin*载脂蛋白具有相似的X射线结构，但却具有完全不同的活性。(3)下一步，我们计划研究整个活性系综中pH和水合作用在视紫红质与转导蛋白相互作用中的具体作用。我们计划将我们的光谱方法(紫外可见、FTIR、定点自旋标记)与渗透压研究相结合，以研究水介导的氢键网络的变化，以及视紫红质激活中跨膜螺旋运动导致的水内流。(4)进一步的研究将揭示多不饱和脂类通过调节膜曲率应力对视紫红质的影响，以及脂类组成如何偏向主动系综机制中的状态分布。我们的计划概括了视紫红质如何启动视觉信号转导的新的多尺度视角，将机械、环境、时间和结构因素联系在一起。了解这些因素如何在不同的长度和时间尺度上相互作用，是理解视紫红质如何实现视觉信号所需的极高保真度的基础。我们强大而新颖的方法将提供重要的见解，可以转移到生物学和药理学中更广泛的GPCR类别。