Structure/Function of Microbial Sensory Rhodopsins

微生物感觉视紫红质的结构/功能

基本信息

批准号：
8638009
负责人：
JOHN LEE SPUDICH
金额：
$ 62.56万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8638009
关键词：
Algae Animals Archaea Automobile Driving Bacteria Bacteriorhodopsins Basic Science Binding Biomedical Research Birth Calcium Channel Cations Cell membrane Cells Chemicals Chlamydomonas reinhardtii Clinical Complex Conflict (Psychology)Cytoplasm Data Development Eukaryota Event Evolution Exhibits Figs - dietary Gene Expression Profile Genetic Techniques Goals Human Hydrogen Hydrogen Bonding In Vitro Ions Knowledge Life Light Mammalian Cell Maps Measurement Measures Mediating Membrane Membrane Potentials Membrane Proteins MicroRNAs Microbial Rhodopsins Molecular Molecular Conformation Molecular Structure Monitor Mus Mutagenesis Neurons Oocytes Optics Phototransduction Play Process Prokaryotic Cells Property Proteins Proton Pump Protons Research Resolution Retinal Retinal Pigments Rhodopsin Roentgen Rays Role Sampling Scaffolding Protein Schiff Bases Sensory Sensory Rhodopsins Side Signal Pathway Signal Transduction Site Spectroscopy, Fourier Transform Infrared Spectrum Analysis Structure System Technology Testing Therapeutic Therapeutic Agents Time Transducers Vesicle Vision base blind brain tissue chromophore conformer design in vivo interest light gated light intensity microbial microorganism multidisciplinary mutant nervous system disorder new technology optogenetics protein function protein protein interaction public health relevance receptor research study restoration sensor sensory rhodopsin I tool transmission process uptake

项目摘要

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.

描述（由申请人提供）：微生物感觉视紫红质，广泛存在于原核生物和单细胞真核生物中的膜嵌入七螺旋光传感器，其信号传导机制显着不同。在古细菌和细菌中，它们通过膜嵌入受体-转导子 (SR-Htr) 复合物中的蛋白质-蛋白质相互作用介导趋光性。相比之下，藻类中的同源感觉视紫红质，通道视紫红质（ChRs），通过使藻类质膜去极化来介导趋光性。光激活提供的优势，即时间精度和光谱工具，已经成为膜蛋白功能和理解进化如何修改现有蛋白质支架以创建新蛋白质功能的微生物视紫红质范例系统。除了基础科学兴趣之外，感觉视紫红质还催生了一种新技术——光遗传学，它利用 ChR 控制动物细胞中的膜电位，从而实现光触发的神经元放电。 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 SR-Htr 复合物的视紫红质亚基，并提供证据表明 ChR 也通过光驱动视紫红质子泵在两个构象异构体之间共享相同的光诱导转换。然而，感觉视紫红质已经进化出新的化学过程，在它们的质子泵祖先中没有发现，以改变构象变化的后果。实验旨在：1）利用我们最近发现的微生物视紫红质构象异构体在黑暗中以稳定形式存在的条件，确定第一个原子分辨率的X射线晶体结构。引诱剂受体SRI-HtrI复合物； 2) 阐明驱避受体 SRII 中视网膜光异构化过程中构象异构体转变和空间触发因素之间的相互作用，它们共同构成了信号传递至 HtrII 的分子内途径； 3) 将我们的 SRI 和 SRII 知识以及多学科工具应用于 ChR。 ChR 的通道活性仅通过活藻类或动物细胞中的光电测量来检测和研究，而 ChR 的浓度不适合光学或分子光谱。我们建议开发一种体外系统，用于纯化 ChR 的光门控通道活性，并阐明该蛋白质的光转导机制。我们对藻类趋光性的研究得出了最终目标，该研究表明 ChR 介导的去极化电流与其在异源系统（例如，藻类）中的活性相比被放大约 1000 倍。神经元。我们提出了一种策略来识别放大成分并测试我们的假设，即非电压门控 Ca2+ 通道是通过与藻类中 ChR 的物理相互作用直接激活的。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使用并实现治疗应用。