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: 7737309
• 负责人: KENNETH J ROTHSCHILD
• 金额: $ 28.44万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7737309
• 关键词: Anabaena; Archaea; Bacteria; Bacteriorhodopsins; Biological Models; Biotechnology; Cell physiology; Cells; Charge; Collaborations; Complex; Crystallization; Cyanobacterium; Development; Environment; Event; Evolution; Family; Fresh Water; GTP-Binding Proteins; Goals; Grant; Green Algae; Ion Channel; Isotope Labeling; Knowledge; Laboratories; Lead; Life; Light; Link; Marines; Measures; Medical center; Membrane; Membrane Proteins; Methods; Microbial Rhodopsins; Modeling; Molecular; Movement; Natronobacterium; Pathway interactions; Peptides; Photons; Physiological; Proteins; Proton Pump; Research; Resolution; Retinal; Rhodopsin; Sensory Rhodopsins; Signal Transduction; Specific qualifier value; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Structure; Techniques; Technology; Testing; Texas; Time; Time Study; Transducers; Universities; Vertebral column; Water; Work; X ray diffraction analysis; X-Ray Diffraction; absorption; base; chromophore; protein protein interaction; protein structure; public health relevance; receptor; response; sensor

DESCRIPTION (provided by applicant): 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. Examples of SRs include SRI and SRII from archaebacteria, which control phototaxis, Anabaena sensory rhodopsin (ASR) from freshwater cyanobacteria, which functions as photochromic sensors, some forms of proteorhodopsin (PR) found in marine bacteria, which control a variety of cellular functions, and the recently discovered channel-rhodopsins (ChRs), which control phototactic and photophobic responses in green algae. In contrast to bacteriorhodopsin (BR), the well-studied light-driven proton pump, most SRs function by transmitting a signal to an associated transducer protein, analogous to the well-known G-proteins in the rhodopsin signaling cascade. Still others, such as ChRs, convey a signal by opening a self-contained light-activated ion channel. 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 protein- protein 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 other SRs, 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 this project we will 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 to, for the first time, 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, ii) rapidly express and isotope label SRs and their transducer complexes using the technology of cell-free expressed nanolipoparticles (NLPs), and iii) measure time-resolved FTIR-differences of SRs in single crystals. 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. Specifi objectives of this project are: PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the signaling mechanism of light activated sensory rhodopsins (SRs). Most SRs function by transmitting a signal to an associated transducer protein. In contrast, channel-rhodopsins convey a signal by opening a self-contained light-activated ion channel. SRs provide an important opportunity to understand how evolution has modified similar membrane protein structures to accomplish very different molecular mechanisms of signaling. In this project we will use an array of advanced IR-based techniques to examine the detailed molecular events which lead to signal activation in SRs including new methods to study these proteins inside living cells.

描述（由申请人提供）：该项目的主要目的是了解光活化感觉视紫红蛋白（SRS）的信号传导机理，这是增长的7螺旋跨膜微生物动蛋白的不断增长的一部分。 Examples of SRs include SRI and SRII from archaebacteria, which control phototaxis, Anabaena sensory rhodopsin (ASR) from freshwater cyanobacteria, which functions as photochromic sensors, some forms of proteorhodopsin (PR) found in marine bacteria, which control a variety of cellular functions, and the recently discovered channel-rhodopsins (ChRs), which control绿藻中的光性和额恐惧症反应。与细菌的淡淡蛋白质（BR）相比，良好的轻驱动质子泵，大多数SRS通过将信号传输到相关的换能蛋白的功能，类似于Rhodopsin信号级联中众所周知的G蛋白。还有其他人（例如CHRS）通过打开一个独立的光激活离子通道传达信号。 SRS信号传导机制分子水平上的详细知识对于理解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中的应用至关重要。对于来自纳第硝杆菌的SRII，与其同源性HTRII透射剂的跨膜部分相关的受体的高分辨率结构揭示了蛋白质蛋白质相互作用的重要分子细节，包括位于界面区域中的接触残留物和内部水分子。然而，到目前为止，X射线衍射尚未揭示​​出将视网膜发色团初始光诱导的异构化与换能器激活相关的分子事件，这可能是由于晶体晶格施加的结构约束所致。在其他SR中，由于结晶和表达的困难，已知信息甚至更少。此外，我们自己和其他研究表明，在天然膜中研究SRS的重要性。理想情况下，需要新技术来研究本机环境（包括内部细胞）中的SR结构变化。在这个项目中，我们将使用一系列基于高级IR的技术，其中一些技术最近在我们的实验室中开发，以检查导致SRS信号激活的详细分子事件。在过去的赠款期间取得了重大进展，导致新的分子细节和SR功能的暂定模型。在拟议的研究中，将通过测量特定残基的结构变化，内部水分子和SR受体转换剂中的肽主链的结构变化来详细测试。拟议的研究的一个独特方面是，可以首次研究完整功能细胞的结构变化，在这些细胞中，可以测量与其他事件直接相关的与其他事件的直接相关性，例如光的和光诱导的电荷运动。 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, ii) rapidly express and isotope label SRs and their transducer complexes using the technology of cell-free expressed nanolipoparticles (NLPs), and iii) measure time-resolved FTIR-differences of单晶中的SR。这项工作将通过与休斯敦大学医学中心的J. Spudich博士的实验室进行密切合作来促进，其实验室为我们目前的SRS贡献了我们当前的大部分知识，而Lawrence Livermore国家实验室的M. Coleman博士则在NLPS中开发了无细胞的技术来表达膜蛋白。该项目的特殊目标是：公共卫生相关性：该项目的目标是了解光激活的感觉视紫红蛋白（SRS）的信号传导机制。大多数SRS通过将信号传输到相关的传感器蛋白来函数。相比之下，通道 - 偶像蛋白通过打开独立的光激活离子通道传达信号。 SRS提供了一个重要的机会来了解进化如何修饰相似的膜蛋白结构，以完成非常不同的信号传导分子机制。在这个项目中，我们将使用一系列高级基于IR的技术来检查详细的分子事件，这些事件导致SRS中的信号激活，包括新方法研究活细胞内的这些蛋白质。