FTIR STUDY OF SIGNAL TRANSDUCTION IN SENSORY RHODOPSINS

这个项目的主要目标是了解光的信号机制。 激活的感觉视紫红质(SRS)，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)。与之形成鲜明对比的是 细菌视紫红质(BR)是一种研究很好的光驱动质子泵，这些SRS通过以下方式发挥作用 将信号传递到相关的转导蛋白，类似于众所周知的G- 视紫红质信号通路中的蛋白质。在分子水平上的详细知识 SRS的信号转导机制对于理解SRS信号转导机制具有重要意义 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。 在来自法老钠细菌的SRII的情况下，其高分辨结构 与其同源HtrII转导的跨膜部分相连的受体已经揭示 蛋白质相互作用的重要分子细节，包括接触 位于界面区域的残留物和内部水分子。然而，到目前为止，X- 射线衍射还没有揭示出连接最初光诱导的分子事件 视网膜生色团的异构化可能是由于感受器的激活 由晶格施加的结构限制。在SRII的情况下，它 调节两种颜色的排斥和吸引反应，甚至更少的信息被知道 由于结晶和表达困难。此外，我们自己和其他方面的研究 论证在当地生理条件下研究SRS的重要性 膜。理想情况下，需要新的技术来研究SR的结构变化 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，用来检查详细的 在SRS中导致信号激活的分子事件。已经取得了重大进展 在过去的授权期内所做的工作导致了新的分子细节和SR的初步模型 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、内部水分子和多肽的结构变化 SR受体-转导复合体中的骨干在亚皮秒的时间尺度上 几秒钟。拟议研究的一个独特方面是第一次有能力研究 这些结构变化在完整的功能细胞中，其中与其他 事件，如趋光性和光致电荷移动，可以被测量。这个 拟议的研究还将受益于我们开发的新方法，以：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及II)快速表达和同位素标记 利用无细胞表达技术实现SRS及其转导复合体 纳米脂微球(NLP)。 这项工作将通过与J博士的实验室的密切合作来促进。 休斯顿德克萨斯大学医学中心的Spudich博士说，他的实验室已经 贡献了我们目前关于SRS的大部分知识，M.Coleman博士在 劳伦斯·利弗莫尔国家实验室，他的团队已经开发出无细胞 在NLP中表达膜蛋白的技术。该项目的具体目标是：

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: