FTIR STUDY OF SIGNAL TRANSDUCTION IN SENSORY RHODOPSINS

感觉视紫红质信号转导的 FTIR 研究

• 批准号: 6873519
• 负责人: KENNETH J ROTHSCHILD
• 金额: $ 29.23万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873519
• 关键词: Raman spectrometry; bioimaging /biomedical imaging; biological signal transduction; cell surface receptors; chimeric proteins; infrared spectrometry; interferometry; membrane proteins; molecular /cellular imaging; phosphorylation; photoactivation; protein structure function; rhodopsin; structural biology; time resolved data

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.

描述(由申请人提供)：本项目的主要目标是了解一类信号转导光受体，即感觉性视紫红质功能的潜在分子机制。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清单。由于感觉性视紫红质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，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。感觉性视紫红质通过将信号传递给相关的甲基受体(HTR)来发挥作用，HTR介导了一系列的磷酸化。在初步研究中，从几种感官视紫质及其突变体中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Schiff碱反离子和其他未知残基的一系列质子化变化。我们已经证明了利用FTIR差示光谱技术研究融合蛋白在体内表达形成的完整的感觉性视紫红质-转导复合体的可行性。这些数据揭示了可能由受体和同源转导的两个核心跨膜螺旋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，在微秒到几秒的时间尺度上检查正常形式和改良形式的感觉性视紫红质及其融合复合体在光激发下发生的分子事件。这项研究将利用时间分辨、偏振和ATR FTIR差光谱以及FT-拉曼光谱。对感光紫质3D晶体的显微FTIR研究将提供捕获的光中间体的X射线结构的信息，这些结构可能会被晶格约束改变。我们还将利用以前应用于细菌视紫红质的先进基因技术，允许在特定位置将同位素标记和非天然氨基酸结合到感官视紫红质及其传感器中。我们详细描述了利用这些方法进行的一系列实验，目的是测试最近提出的几种信号转导机制。