FTIR Study of Signal Transduction in Sensory Rhodopsins

描述（由申请人提供）：本项目的主要目的是了解光激活感觉视紫红质（SR）的信号传导机制，SR是7-螺旋跨膜微生物视紫红质家族的一部分。SR的实例包括来自古细菌的SRI和SRII，其控制趋光性，来自淡水蓝藻的鱼腥藻感觉视紫红质（ASR），其用作光致变色传感器，在海洋细菌中发现的某些形式的蛋白视紫红质（PR），其控制多种细胞功能，以及最近发现的通道视紫红质（ChR），其控制绿色藻类中的趋光性和疏水性反应。与细菌视紫红质（BR）（研究充分的光驱动质子泵）相反，大多数SR通过将信号传递到相关的转导蛋白来起作用，类似于视紫红质信号级联中众所周知的G蛋白。还有一些，如ChRs，通过打开一个独立的光激活离子通道来传递信号。在分子水平上对SRs信号传导机制的详细了解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的应用具有重要意义。在SRII的情况下，从嗜盐杆菌pharaonis，连接到跨膜部分的同源HtrII转换器的受体的高分辨率结构揭示了重要的分子细节的蛋白质-蛋白质相互作用，包括接触残基和内部水分子位于界面区域。然而，到目前为止，X-射线衍射还没有揭示的分子事件连接初始光诱导异构化的视网膜生色团的激活的换能器，可能是由于晶格的结构约束。在其他SR的情况下，甚至更少的信息是已知的，由于结晶和表达的困难。此外，我们自己和其他研究表明，在天然膜的生理条件下研究SR的重要性。理想情况下，需要新的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IR的技术，其中一些是最近在我们的实验室开发的，以检查详细的分子事件，导致信号激活的SR。在过去的资助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，这些模型将通过在亚皮秒至秒的时间尺度上测量SR受体-换能器复合物中特定残基、内部水分子和肽骨架的结构变化来详细测试。拟议研究的一个独特方面是能够首次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（如趋光性和光诱导电荷运动）的直接相关性。拟议的研究也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法测量蛋白质及其内部水分子的亚皮秒结构变化，ii）使用无细胞表达纳米脂质体（NLP）技术快速表达和同位素标记SR及其换能器复合物，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的J. Spudich博士的实验室的密切合作来促进，他的实验室为我们提供了关于SR的大部分现有知识，而M.劳伦斯利弗莫尔国家实验室的科尔曼说，他的小组已经开发出无细胞技术来表达NLP中的膜蛋白。本项目的具体目标是：公共卫生相关性：本项目的目标是了解光激活的感觉视紫红质（SR）的信号机制。大多数SR通过将信号传递到相关的转导蛋白来发挥功能。相反，通道视紫红质通过打开自含的光激活离子通道来传递信号。SR提供了一个重要的机会，了解进化如何修改类似的膜蛋白结构，以实现非常不同的分子机制的信号。在这个项目中，我们将使用一系列先进的基于IR的技术来研究导致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。

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.