FTIR Study of Signal Transduction in Sensory Rhodopsins

描述(申请人提供)：这个项目的主要目标是了解光激活的感觉性视紫红质(SRS)的信号机制，SRS是不断增长的7-螺旋跨膜微生物视紫红质家族的一部分。SRS的例子包括控制趋光性的古细菌的SRI和SRII，作为光致变色传感器的淡水蓝藻的鱼腥藻感觉视紫红质(ASR)，在海洋细菌中发现的某些形式的蛋白视紫红质(PR)，它控制各种细胞功能，以及最近发现的通道视紫红质(CHR)，它控制绿藻的趋光性和畏光反应。与光驱动质子泵细菌视紫红质(BR)不同，大多数SRS通过向相关的转导蛋白传递信号来发挥作用，类似于视紫红质信号级联中众所周知的G蛋白。还有一些，如CHR，通过打开一个独立的光激活离子通道来传递信号。在分子水平上详细了解SRS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的应用具有重要意义。以法老钠杆菌的SRII为例，与其同源HtrII转导蛋白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，包括位于界面区域的接触残基和内部水分子。然而，到目前为止，X射线衍射还没有揭示出将视网膜发色团最初的光诱导异构化与转导激活联系起来的分子事件，这可能是由于晶格的结构限制。在其他SRS的情况下，由于结晶和表达的困难，所知的信息甚至更少。此外，我们自己的研究和其他研究证明了在天然膜的生理条件下研究SRS的重要性。理想情况下，需要新的技术来研究自然环境中SR结构的变化，甚至包括细胞内的变化。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，来研究导致SRS中信号激活的详细分子事件。在过去的资助期间，已经取得了重大进展，导致了SR功能的新的分子细节和初步模型。在拟议的研究中，这些模型将通过在亚皮秒到几秒的时间尺度上测量SR受体-转导复合体中特定残基、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，第一次能够研究完整功能细胞的这些结构变化，其中可以测量到与其他事件直接相关的事件，如趋光性和光致电荷移动。我们的研究还将得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)使用无细胞表达纳米脂肪颗粒(NLP)技术快速表达和同位素标记SRS及其传感器复合体，以及III)测量单晶中SRS的时间分辨FTIR差。这项工作将通过与休斯顿德克萨斯大学医学中心的J.Spudich博士和劳伦斯·利弗莫尔国家实验室的M.Coleman博士的密切合作来促进，前者的实验室贡献了我们目前关于SRS的大部分知识，后者的团队开发了在NLP中表达膜蛋白的无细胞技术。该项目的具体目标是：与公共健康相关：该项目的目标是了解光激活的感觉性视紫红质(SRS)的信号机制。大多数SRS通过将信号传递给相关的转导蛋白来发挥作用。相比之下，通道-视紫红质通过打开一个独立的光激活离子通道来传递信号。SRS为理解进化如何改变相似的膜蛋白结构以实现完全不同的信号分子机制提供了一个重要的机会。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SRS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.