MEMBRANE BASIS OF VISUAL EXCITATION

描述（由申请人提供）：我们的研究旨在建立原子和膜水平的事件如何在统一的多尺度框架中触发视紫红质的视觉信号转导，对生物信号传导具有广泛的影响。理解G蛋白偶联受体（GPCR）激活的高度影响是很好的赞赏。然而，我们的理解仍然存在许多差距，无论是与视紫红质，以及其他家庭A GPCR，视紫红质是一个非常重要的原型。在这里，我们计划通过结合磁共振（固态2 H和13 C NMR），傅里叶变换红外（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制特征。我们的新假说的视紫红质激活制定的因素，通过一个积极的合奏，推动一个进程的瞬态构象substates：释放视网膜应变，视网膜特异性蛋白质动力学，水合作用的变化，pH催化，动态G-蛋白偶联，和膜应力由于多不饱和脂质组合物。(1)我们的新型固态2 H和13 C NMR技术的应用将揭示如何通过视网膜异构化和弛豫释放构象应变解锁活性视紫红质（Meta-II）状态。2 H和13 C固态NMR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力学结构。(2)通过2 H和13 C NMR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结合的光谱与分子动力学（MD）模拟的结果。局部视网膜流动性将涉及大规模的蛋白质动力学，涉及跨膜螺旋的波动。值得注意的是，这项工作将解决模糊的X射线结构数据与在更多的生理温度下获得的结果。我们的研究将决定为什么活跃的Meta-II视紫红质和无配体的Opsin* apoprotein具有相似的X射线结构，但完全不同的活动的问题。(3)接下来，我们计划调查的具体作用f pH值和水合作用在整个活跃的合奏有关视紫红质的相互作用与transducin。我们计划联合收割机结合我们的光谱方法（紫外可见，FTIR，定点自旋标记）与渗透压的研究，以调查水介导的氢键网络的变化，以及一个戏剧性的水流入由于视紫红质激活跨膜螺旋运动。(4)进一步的研究将揭示多不饱和脂质通过调节膜曲率应力对视紫红质的影响，以及脂质成分如何使活性系综机制中的状态分布偏置。我们的计划包含了一个新的多尺度视图视紫红质如何启动视觉信号转导，捆绑在一起的机械，环境，时间和结构因素。了解这些因素如何在不同的长度和时间尺度上相互作用，对于理解视紫红质如何实现视觉信号所需的极高保真度至关重要。我们强大而新颖的方法将提供重要的见解，这些见解可以转移到生物学和药理学中更广泛的GPCR类别。

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.