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）：该项目的主要目标是了解光激活感觉视紫红质 (SR) 的信号传导机制，SR 是不断增长的 7 螺旋跨膜微生物视紫红质家族的一部分。 SR 的例子包括来自古细菌的 SRI 和 SRII，它们控制趋光性；来自淡水蓝细菌的鱼腥藻感觉视紫红质 (ASR)，它充当光致变色传感器；在海洋细菌中发现的某些形式的蛋白视紫红质 (PR)，它控制多种细胞功能，以及最近发现的通道视紫红质（ChRs），它控制绿藻的趋光和畏光反应。与细菌视紫红质 (BR)（经过充分研究的光驱动质子泵）相反，大多数 SR 通过将信号传输到相关的转导蛋白来发挥作用，类似于视紫红质信号级联中众所周知的 G 蛋白。还有一些，例如 ChR，通过打开独立的光激活离子通道来传递信号。在分子水平上详细了解SR信号传导机制对于理解各种基于膜蛋白的细胞过程具有重要意义，并在生物技术和生物医学领域具有应用价值。就来自法老嗜盐杆菌的 SRII 而言，与其同源 HtrII 转导器的跨膜部分相连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。然而，到目前为止，X射线衍射尚未揭示​​将视网膜发色团的初始光诱导异构化与传感器激活联系起来的分子事件，这可能是由于晶格施加的结构限制。对于其他 SR，由于结晶和表达的困难，已知的信息甚至更少。此外，我们自己和其他研究证明了在天然膜的生理条件下研究 SR 的重要性。理想情况下，需要新技术来研究天然环境中的 SR 结构变化，甚至包括细胞内部。在这个项目中，我们将使用一系列先进的基于红外的技术（其中一些是我们实验室最近开发的）来检查导致 SR 中信号激活的详细分子事件。在过去的资助期内，我们取得了重大进展，产生了新的分子细节和 SR 功能的初步模型。在拟议的研究中，这些模型将通过在亚皮秒到秒的时间尺度上测量 SR 受体-转导复合物中特定残基、内部水分子和肽主链的结构变化来进行详细测试。拟议研究的一个独特方面是首次能够研究完整功能细胞中的这些结构变化，其中可以测量与其他事件（例如趋光性和光致电荷运动）的直接相关性。拟议的研究还将受益于我们开发的新方法：i) 使用先进的超快时间分辨红外光谱测量蛋白质及其内部水分子的亚皮秒结构变化，ii) 快速表达和同位素标记 SR 及其传感器复合物使用无细胞表达纳米脂质颗粒 (NLP) 技术，以及 iii) 测量单晶中 SR 的时间分辨 FTIR 差异。这项工作将通过与休斯顿德克萨斯大学医学中心 J. Spudich 博士和劳伦斯利弗莫尔国家实验室的 M. Coleman 博士的实验室的密切合作来促进，该实验室贡献了我们目前有关 SR 的大部分知识。 Laboratories 的团队开发了在 NLP 中表达膜蛋白的无细胞技术。该项目的具体目标是： 公共健康相关性：该项目的目标是了解光激活感觉视紫红质 (SR) 的信号传导机制。大多数 SR 通过将信号传输到相关的转导蛋白来发挥作用。相反，通道视紫红质通过打开独立的光激活离子通道来传递信号。 SR 提供​​了一个重要的机会来了解进化如何改变相似的膜蛋白结构以实现截然不同的信号传导分子机制。在这个项目中，我们将使用一系列先进的基于红外的技术来检查导致 SR 信号激活的详细分子事件，包括研究活细胞内这些蛋白质的新方法。