Structure/Function of Microbial Sensory Rhodopsins

描述(申请人提供)：微生物感觉性视紫红质，一种广泛存在于原核生物和单细胞真核生物中的膜嵌入型7螺旋光传感器，其信号机制非常不同。在古生菌和细菌中，它们通过膜嵌入型受体-转导器(SR-HTR)复合体中的蛋白质-蛋白质相互作用来调节趋光性。相反，藻类中的同源感官视紫红质，通道视紫红质(CHRs)，通过去极化藻质膜来调节趋光性。光激活提供的优势，即时间精度和光谱工具，使微生物视紫红质成为膜蛋白功能的范例系统，并理解进化如何修改现有的蛋白质支架以创造新的蛋白质功能。除了基本的科学兴趣外，感官视紫红质还催生了一项新技术--光遗传学，它使用CHRs控制动物细胞的膜电位，使光触发的神经元放电成为可能。CHRS已成为神经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性视紫红质机制的基本原理。我们已经确定了SR-HTR复合体的视紫红质亚单位，并提供了证据表明，CHR也在具有光驱动视紫红质质子泵的两个构象之间共享相同的光诱导转换。然而，感觉性视紫红质已经进化出了它们的质子泵祖先所没有的新的化学过程，以改变构象变化的后果。实验的目的是：1)利用我们最近发现的条件，确定难以捉摸的瞬时光诱导的微生物视紫红质构象的原子分辨X射线晶体结构，在黑暗中，这种构象在引诱剂中以稳定的形式存在 受体SRI-HtrI复合体；2)在排斥受体SRII中阐明视网膜光异构化过程中构象转换和空间触发之间的相互作用，这两者共同构成了信号传递到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CHRS。CHRs的通道活性仅通过在活的藻类或动物细胞中进行光电测量来检测和研究，其浓度不适用于光学或分子光谱。我们建议建立一个体外系统来检测纯化的CHRS的光门通道活性，并阐明该蛋白的光传导机制。最后一个目标来自我们对藻类趋光性的研究，该研究证实，与其在异源系统(如神经元)中的活性相比，ChR介导的去极化电流放大了约1000倍。我们提出了一种确定放大成分(S)的策略，并验证了我们的假设，即非电压门控钙通道通过与藻类CHRs的物理相互作用直接激活。除了回答基本的机制问题外，这些实验还有可能对光遗传学产生重大影响，加强这项技术在研究中的使用，并使治疗应用成为可能。

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.