FTIR STUDY OF SIGNAL TRANSDUCTION IN SENSORY RHODOPSINS

感觉视紫红质信号转导的 FTIR 研究

• 批准号: 7342112
• 负责人: KENNETH J ROTHSCHILD
• 金额: $ 22.97万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7342112
• 关键词: Active Biological Transport; Amino Acids; Anabaena; Animals; Archaea; Bacteriorhodopsins; Biological Models; Chimeric Proteins; Class; Communication; Complex; Crystallography; Cyanobacterium; Data; Eubacterium; Eukaryota; Eukaryotic Cell; Event; Family; Feasibility Studies; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic Techniques; Goals; Halobacterium salinarium; Helix (Snails); Integral Membrane Protein; Isotope Labeling; Laboratories; Light; Mediating; Membrane; Membrane Proteins; Methods; Microbial Rhodopsins; Microscopic; Modeling; Molecular; Natronobacterium; Neurospora crassa; Peptides; Phosphorylation; Proteins; Protons; Raman Spectrum Analysis; Research; Resolution; Retinal; Retinal Pigments; Rhodopsin; Roentgen Rays; Schiff Bases; Sensory; Sensory Rhodopsins; Series; Signal Transduction; Signal Transduction Pathway; Site; Site-Directed Mutagenesis; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Structural Models; Structure; Techniques; Testing; Time; Transducers; Vertebral column; Water; Work; absorption; base; chromophore; deprotonation; ear helix; experience; fungus; in vivo; mutant; protonation; receptor; research study; sensory rhodopsin I; transmission process

The primary objective of this project is to understand the signaling mechanism of light activated sensory rhodopsins (SRs), part of the growing family of 7-helix transmembrane microbial rhodopsins. The focus of this research will be on two key microbial rhodopsins, sensory rhodopsins I (SRI) and sensory rhodopsin II (SRII). In contrast to bacteriorhodopsin (BR), the well-studied light-driven proton pump, these SRs function by transmitting a signal to an associated transducer protein, analogous to the well-known G- proteins in the rhodopsin signaling cascade. Detailed knowledge at the molecular level of the signaling mechanisms of SRs would be of great significance for understanding a variety of membrane protein-based cellular processes as well as have applications in the field of biotechnology and biomedicine. In the case of SRII from Natronobacterium pharaonis, the high-resolution structure of the receptor linked to the transmembrane part of its cognate HtrII transducer has revealed important molecular details of the proteinprotein interactions, including the contact residues and internal water molecules located in the interface region. However, so far X- ray diffraction has not revealed the molecular events connecting the initial light-induced isomerization of the retinal chromophore to the activation of the transducer, possibly due to structural constraints imposed by the crystal lattice. In the case of SRII, which mediates a two-color repellent and attractant response, even less information is known due to difficulties of crystallization and expression. In addition, our own and other studies demonstrate the importance of studying SRs under physiological conditions in native membranes. Ideally, new techniques are needed for studying SR structural changes in a native environment, including even the inside the cell. In the revised project we will continue to use an array of advanced IR-based techniques, some of which have recently been developed in our laboratory, to examine the detailed molecular events which lead to signal activation in SRs. Significant progress has been made in the past grant period leading to new molecular details and tentative models of SR function. In the proposed research, these models will be tested in detail by measuring structural changes of specific residues, internal water molecules, and the peptide backbone in SR receptor-transducer complexes on a time-scale of sub-picoseconds to seconds. A unique aspect of the proposed studies is the ability, for the first time, to study these structural changes in intact functioning cells where direct correlation with other events, such as phototaxis and photoinduced charge movements, can be measured. The proposed studies will also benefit from our development of new methods to: i) measure sub-picosecond structural changes in the protein and its internal water molecules using advanced ultrafast time-resolved IR spectroscopy and ii) rapidly express and isotope label SRs and their transducer complexes using the technology of cell-free expressed nanolipoparticles (NLPs). This work will be facilitated by close collaborations with the laboratories of Dr. J. Spudich at the University of Texas Medical Center, Houston, whose laboratory has contributed much of our current knowledge about SRs, and Dr. M. Coleman at the Lawrence Livermore National Laboratories, whose group has developed cell-free techniques to express membrane proteins in NLPs. Specific objectives of this project are:

该项目的主要目的是了解光的信号传导机制 活化的感觉视opsin（SRS），这是增长的7螺旋跨膜家族的一部分 微生物视紫红蛋白。这项研究的重点将放在两个关键的微生物视紫红蛋白上，即 感觉视紫红蛋白I（SRI）和感觉视紫红质II（SRII）。与 细菌二红蛋白（BR），良好的轻驱动质子泵，这些SRS功能 将信号传输到相关的传感器蛋白，类似于众所周知的g- 视紫红质信号级联反应中的蛋白质。分子水平的详细知识 SR的信号传导机制对于理解A具有重要意义 各种基于膜蛋白的细胞过程，以及在 生物技术和生物医学领域。 对于来自纳特隆杆菌Pharaonis的SRII，高分辨率结构 与其同源HTRII透射剂的跨膜部分相关的受体已揭示 蛋白质蛋白相互作用的重要分子细节，包括接触 残留物和内部水分子位于界面区域。但是，到目前为止x- 射线衍射尚未揭示​​连接初始光诱导的分子事件 视网膜发色团的异构化与换能器的激活，可能是由于 晶格施加的结构约束。以Srii为例 介导两种驱动力和引人入胜的响应，已知信息甚至更少 由于结晶和表达的困难。此外，我们自己和其他研究 证明在生理条件下研究SRS的重要性 膜。理想情况下，需要新技术来研究A中的SR结构变化 本地环境，包括内部的细胞。 在修订的项目中，我们将继续使用一系列基于IR的高级技术， 其中一些最近是在我们的实验室中开发的，以检查详细的 导致SRS信号激活的分子事件。取得了重大进展 在过去的赠款期间制造，导致新的分子细节和SR的暂定模型 功能。在拟议的研究中，将通过测量来详细测试这些模型 特定残基，内部水分子和肽的结构变化 SR受体转导仪复合物中的主链在亚皮秒的时间尺度上 秒。拟议研究的一个独特方面是首次研究的能力 完整功能细胞中的这些结构变化，其中直接与其他相关 可以测量事件，例如光触电和光诱导的电荷运动。这 拟议的研究还将受益于我们的新方法的开发：i）测量 使用蛋白质及其内部水分子的次秒结构变化 晚期超快时间分辨的红外光谱和II）快速表达和同位素标签 SRS及其传感器复合物使用无细胞表达的技术 纳米颗粒（NLP）。 与J.博士的实验室密切合作，将促进这项工作。 休斯顿德克萨斯大学医学中心的Spudich，其实验室已有 我们目前对SRS的知识有所贡献，M。Coleman博士在 劳伦斯·利弗莫尔国家实验室，其小组不含细胞 在NLP中表达膜蛋白的技术。该项目的具体目标是：