FTIR STUDY OF SIGNAL TRANSDUCTION IN SENSORY RHODOPSINS

描述（由申请人提供）：该项目的主要目标是了解一类被称为感觉视紫红质的信号转导光受体功能的分子机制。其中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清单。由于其与细菌视紫红质的密切关系，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一个很好的模型系统。它们也与真细菌中的趋化受体密切相关。感觉视紫红质通过向相关的甲基接受换能器（Htr）传递信号，介导磷酸化级联发挥作用。在初步研究中，从几种感觉紫红质及其突变体获得了静态和时间分辨的FTIR差谱，揭示了构象变化的存在，包括涉及希夫碱反离子和其他未识别残基的一系列质子化变化。我们已经证明了使用FTIR差分光谱来研究由融合蛋白在体内表达形成的完整感觉视紫红质-传感器复合物的可行性。这些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相互作用产生的信号。在提出的研究中，一系列基于红外的技术将用于在微秒到秒的时间尺度上检查正常和修饰形式的感觉视紫红质及其融合复合物在光激发下发生的分子事件。本研究将利用时间分辨、极化、ATR傅里叶变换红外差分光谱以及傅里叶变换拉曼光谱。对感觉紫红质三维晶体的微观FTIR研究将提供捕获光中间体的x射线衍生结构的信息，这些结构可能受到晶格约束的改变。我们还将利用先前应用于细菌视紫红质的先进遗传技术，使同位素标记和非天然氨基酸在特定位点被纳入感觉视紫红质及其传感器中。我们详细描述了一系列利用这些方法的实验，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.