MEMBRANE BASIS OF VISUAL EXCITATION

描述（由申请人提供）：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中触发视紫红质的视觉信号转导，这对生物信号传导具有广泛的影响。了解g蛋白偶联受体（GPCR）激活的高影响是值得赞赏的。然而，我们对视紫红质以及视紫红质是非常重要的原型的其他A家族gpcr的理解仍然存在许多差距。在这里，我们计划通过结合磁共振（固态2H和13C 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。我们关于视紫红质活化的新假设是根据驱动瞬态构象亚态进展的因素制定的，这些因素通过一个活跃的集合：视网膜应变的释放，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，以及由于多不饱和脂质组成的膜应力。(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质（Meta-II）状态。2H和13C固体核磁共振的角度和距离约束将阐明视网膜活性亚态向活性Meta-II形式发展的动力学结构。(2)通过2H和13C核磁共振弛豫研究，将光谱学结果与分子动力学（MD）模拟相结合，研究视网膜局部动力学的变化与视紫红质激活运动的相关性。局部视网膜移动将与涉及跨膜螺旋波动的大规模蛋白质动力学有关。值得注意的是，这项工作将解决在生理温度下获得的模糊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视蛋白*载子蛋白具有相似的x射线结构，但活性完全不同。(3)接下来，我们计划研究pH和水合作用在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（uv -可见，FTIR，位点定向自旋标记）与渗透压研究结合起来，研究水介导的氢键网络的变化，以及由于紫红质激活的跨膜螺旋运动而引起的大量水流入。(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，以及在主动系综机制中，脂质组成如何影响状态分布。我们的计划包含了一个新的多尺度视角，即视紫红质如何启动视觉信号转导，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。我们稳健而新颖的方法将提供重要的见解，可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.