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:

该项目的主要目标是了解光的信号传导机制 激活的感觉视紫红质 (SR)，是不断增长的 7 螺旋跨膜家族的一部分 微生物视紫红质。这项研究的重点将是两种关键的微生物视紫红质， 感觉视紫红质 I (SRI) 和感觉视紫红质 II (SRII)。相比之下 细菌视紫红质 (BR) 是一种经过充分研究的光驱动质子泵，这些 SR 的功能是通过 将信号传输至相关的转导蛋白，类似于众所周知的 G- 视紫红质信号级联中的蛋白质。分子水平的详细知识 SR 的信号机制对于理解 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。 对于来自法老嗜盐杆菌的 SRII，其高分辨率结构 与其同源 HtrII 传感器跨膜部分相连的受体已揭示 蛋白质蛋白质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。然而，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导致传感器的激活，可能是由于 晶格施加的结构约束。就 SRII 而言， 介导两种颜色的驱避和引诱反应，已知的信息甚至更少 由于结晶和表达困难。此外，我们自己的研究和其他研究 证明在本地人的生理条件下研究 SR 的重要性 膜。理想情况下，需要新技术来研究 SR 结构变化 原生环境，甚至包括细胞内部。 在修订后的项目中，我们将继续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，以检查详细的 导致 SR 信号激活的分子事件。已取得重大进展 在过去的资助期内取得了新的分子细节和 SR 的暂定模型 功能。在拟议的研究中，这些模型将通过测量进行详细测试 特定残基、内部水分子和肽的结构变化 SR受体-换能器复合物中的主干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能够研究 完整功能细胞的这些结构变化与其他细胞直接相关 可以测量趋光性和光诱导电荷运动等事件。这 拟议的研究也将受益于我们开发的新方法：i) 测量 蛋白质及其内部水分子的亚皮秒结构变化 先进的超快时间分辨红外光谱和 ii) 快速表达和同位素标记 采用无细胞表达技术的 SR 及其转导复合物 纳米脂质颗粒（NLP）。 与 J. 博士实验室的密切合作将促进这项工作。 休斯敦得克萨斯大学医学中心的斯普迪奇 (Spudich) 的实验室 M. Coleman 博士贡献了我们当前有关 SR 的大部分知识。 劳伦斯利弗莫尔国家实验室，其团队开发出无细胞 在 NLP 中表达膜蛋白的技术。该项目的具体目标是：