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一类称为感觉视紫红质的信号转导光受体的分子机制。这些包括来自古细菌的感觉视紫红质I和II（SRI和SRII）以及最近在真细菌和真核生物中发现的快速增长的感觉视紫红质。由于它们与细菌视紫红质的关系密切，与视紫红质G蛋白偶联受体的相似性以及高分辨率结构模型的可用性，感觉视紫红质为研究信号转导和膜蛋白相互作用提供了极好的模型系统。它们也与真细菌中的趋化性受体密切相关。感觉视紫红质通过将信号传递到介导磷酸化级联反应的相关甲基接受换能器（Htr）来起作用。在初步研究中，静态和时间分辨的FTIR差光谱已获得从几个感官视紫红质和它们的突变体，揭示了存在的构象变化，包括一系列的质子化变化涉及的席夫碱countryside和其他身份不明的残基。我们已经证明了使用FTIR差光谱研究完整的感觉视紫红质-传感器复合物的可行性，该复合物是由融合蛋白在体内表达形成的。该数据揭示了可能由受体和同源换能器的两个核心跨膜螺旋的相互作用产生的信号。在拟议的研究中，将使用一系列基于红外线的技术来检查在微秒到秒的时间尺度上光激发正常和修饰形式的感觉视紫红质及其融合复合物时发生的分子事件。这项研究将利用时间分辨，偏振，ATR FTIR-差分光谱以及FT-拉曼光谱。显微红外光谱的三维晶体的感官视紫红质的研究将提供信息的X-射线衍生结构的捕获的光中间体，这可能会改变晶格约束。我们还将利用以前应用于细菌视紫红质的先进遗传技术，该技术允许同位素标记和非天然氨基酸在特定位点掺入到感觉视紫红质及其换能器中。我们详细描述了一系列的实验，利用这些方法，旨在测试最近提出的几种信号转导机制。

DESCRIPTION (provided by applicant): The primary objective of this project is to understand the molecular mechanisms underlying the function of a class of signal transducing light receptors known as sensory rhodopsins. These include sensory rhodopsin I and II (SRI and SRII) from archaebacteria and a rapidly growing list of recently discovered sensory rhodopsins in eubacteria and eukaryotes. Because of their close relationship to bacteriorhodopsin, similarity to rhodopsin G-protein coupled receptors and the availability of high resolution structural models, the sensory rhodopsins provide an excellent model system for studying signal transduction and membrane protein interactions. They are also closely related to chemotactic receptors in eubacteria. Sensory rhodopsins function by transmitting a signal to an associated methyl-accepting transducer (Htr) which mediates a phosphorylation cascade. In preliminary studies, static and time-resolved FTIR difference spectra have been obtained from several sensory rhodopsins and their mutants which reveal the presence of conformational changes including a sequence of protonation changes involving the Schiff base counterion and other unidentified residues. We have demonstrated the feasibility of using FTIR difference spectroscopy to study intact sensory rhodopsin-transducer complexes which are formed by in vivo expression of a fusion protein. This data reveals signals which may arise from the interaction of the receptor and the two core transmembrane helices of the cognate transducer. In the proposed research, an array of infrared-based techniques will be used to examine molecular events occurring upon light excitation of normal and modified forms of sensory rhodopsins and their fusion complexes on the time-scale of microseconds to seconds. This research will utilize time-resolved, polarized, and ATR FTIR-difference spectroscopy as well as FT-Raman spectroscopy. Microscopic FTIR studies on 3D crystals of sensory rhodopsins will provide information on X-ray derived structures of trapped photointermediates which may be altered by lattice constraints. We will also utilize advanced genetic techniques previously applied to bacteriorhodopsin, which allow isotope labels and non-native amino acid to be incorporated at specific sites into sensory rhodopsins and their transducers. We describe in detail a series of experiments utilizing these methods aimed at testing several mechanisms of signal transduction which have been recently proposed.