Structure/Function of Microbial Sensory Rhodopsins

描述（由申请人提供）：微生物感觉视紫红质是一种广泛存在于原核生物和单细胞真核生物中的膜包埋7-螺旋光传感器，其信号传导机制非常多样。在古细菌和细菌中，它们通过膜包埋受体-转导子（SR-Htr）复合物中的蛋白质-蛋白质相互作用来介导趋光性。相反，藻类中的同源感觉视紫红质，通道视紫红质（ChRs），介导趋光性通过去极化藻类质膜。光激活提供的优势，即时间精度和光谱工具，使微生物视紫红质范式系统膜蛋白功能和理解如何进化修改现有的蛋白质支架，创造新的蛋白质功能。除了它们的基本科学兴趣之外，感觉视紫红质还产生了一种新技术，即光遗传学，它使用ChRs来控制动物细胞的膜电位，从而实现光触发神经元放电。ChR已成为神经系统疾病研究中广泛使用的研究工具，并提供作为治疗剂的前景。我们的目标是在原子结构/分子功能水平上阐明微生物感觉视紫红质机制的基本原理。我们已经建立了SR-Htr复合物的视紫红质亚基，并提供证据表明，ChRs以及，共享光驱动的视紫红质质子泵的两个构象之间的相同的光诱导转换。然而，感觉视紫红质已经进化出了新的化学过程，在它们的质子泵祖先中没有发现，以改变构象变化的结果。实验旨在：1）通过利用我们最近发现的条件，确定微生物视紫红质的难以捉摸的瞬态光诱导构象的第一原子分辨率X射线晶体结构，其中该构象在黑暗中以稳定形式存在于引诱剂中。 受体SRI-HtrI复合物; 2）阐明在驱避剂受体SRII中构象转换和在视黄醛的光异构化期间的空间触发之间的相互作用，构象转换和空间触发一起构成信号传递到HtrII的分子内途径; 3）将我们对SRI和SRII的知识以及多学科工具应用于ChR。通道活动的ChRs已被检测和研究专门通过光电测量在活的藻类或动物细胞，在浓度的ChRs不服从光学或分子光谱。我们建议开发一个体外系统的光门控通道活性的纯化的ChRs和阐明蛋白质的光转导机制。最后一个目标来自于我们对藻类趋光性的研究，该研究确定了ChR介导的去极化电流与其在异源系统（例如神经元）中的活性相比放大了约1000倍。我们提出了一种策略，以确定放大组件（S）和测试我们的假设，即非电压门控钙通道直接激活的物理相互作用与叶绿素在藻类。除了回答基本的机制问题外，这些实验还可能对光遗传学产生重大影响，增强该技术在研究中的应用并实现治疗应用。

DESCRIPTION (provided by applicant): Microbial sensory rhodopsins, membrane-embedded 7-helix light-sensors widespread among prokaryotes and unicellular eukaryotes, are remarkably diverse in their signaling mechanisms. In archaea and bacteria they mediate phototaxis by protein-protein interaction in membrane-embedded receptor-transducer (SR-Htr) complexes. In contrast, homologous sensory rhodopsins in algae, channelrhodopsins (ChRs), mediate phototaxis by depolarizing the algal plasma membrane. The advantages provided by light activation, namely temporal precision and spectroscopic tools, have made microbial rhodopsins paradigm systems for membrane protein function and for understanding how evolution modifies existing protein scaffolds to create new protein functions. In addition to their basic science interest, sensory rhodopsins have given birth to a new technology, optogenetics, which uses ChRs to control membrane potential in animal cells, enabling light-triggered neuron firing. ChRs have become widely used research tools in neurological disease research and offer promise as therapeutic agents. Our goal is to elucidate the underlying principles of microbial sensory rhodopsin mechanisms at the level of atomic structure/molecular function. We have established that the rhodopsin subunits of SR-Htr complexes, and provide evidence that ChRs as well, share the same light-induced conversion between two conformers with light-driven rhodopsin proton pumps. However, sensory rhodopsins have evolved new chemical processes, not found in their proton pump ancestors, to alter the consequences of the conformational change. Experiments are designed to: 1) determine the first atomic-resolution X-ray crystal structure of the elusive transient light-induced conformer of microbial rhodopsins by exploiting our recent finding of conditions in which this conformer exists as a stable form in the dark in the attractant receptor SRI- HtrI complex; 2) to elucidate in the repellent receptor SRII the interplay between the conformer transition and a steric trigger during photoisomerization of retinal that together comprise the intramolecular pathway of signal transfer to HtrII; 3) to apply our knowledge of SRI and SRII and multidisciplinary tools to the ChRs. The channel activity of ChRs has been detected and investigated exclusively by photoelectric measurements in living algae or animal cells, at concentrations of ChRs not amenable to optical or molecular spectroscopy. We propose to develop an in vitro system for light-gated channel activity of purified ChRs and elucidate the protein's phototransduction mechanism. A final aim follows from our studies of phototaxis in algae which establish that ChR-mediated depolarizing currents are amplified ~1000-fold compared to their activity in heterologous systems, e.g. neurons. We propose a strategy to identify the amplification component(s) and test our hypothesis that non-voltage-gated Ca2+ channels are directly activated by physical interaction with ChRs in the algae. In addition to answering basic mechanistic questions, these experiments have the potential for major impact on optogenetics, enhancing use of this technology in research and enabling therapeutic applications.