Structure/Function of Microbial Sensory Rhodopsins

描述（由申请人提供）：微生物感觉紫红质是广泛存在于原核生物和单细胞真核生物中的膜嵌入7-螺旋光传感器，其信号传导机制非常不同。在古细菌和细菌中，它们通过膜嵌入受体-换能器（SR-Htr）复合物中的蛋白-蛋白相互作用介导趋光性。相比之下，藻类中同源的感觉紫红质，通道紫红质（ChRs），通过使藻质膜去极化来介导趋光性。光激活所提供的优势，即时间精度和光谱工具，使微生物视紫红质成为膜蛋白功能的范例系统，并有助于理解进化如何改变现有的蛋白质支架以创造新的蛋白质功能。除了它们的基础科学兴趣外，感觉视紫红质还催生了一项新技术——光遗传学，该技术利用ChRs控制动物细胞的膜电位，使光触发神经元放电。ChRs已成为神经系统疾病研究中广泛使用的研究工具，并有望成为治疗药物。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。我们已经确定了SR-Htr复合物的视紫红质亚基，并提供证据表明，在光驱动视紫红质质子泵的情况下，ChRs在两个构象之间具有相同的光诱导转换。然而，感觉视紫红质已经进化出新的化学过程，这是它们的质子泵祖先所没有的，以改变构象变化的后果。实验旨在：1)通过利用我们最近发现的在黑暗中以稳定形式存在于引诱剂中的条件，确定微生物紫红质难以捉摸的瞬态光诱导构象的第一个原子分辨率x射线晶体结构

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.