FTIR STUDY OF SIGNAL TRANSDUCTION IN SENSORY RHODOPSINS

感觉视紫红质信号转导的 FTIR 研究

基本信息

批准号：
7175444
负责人：
KENNETH J ROTHSCHILD
金额：
$ 22.97万
依托单位：
BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
依托单位国家：
美国
项目类别：
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/7175444
关键词：
Active Biological Transport Amino Acids Anabaena Animals Archaea Bacteriorhodopsins Biological Models Chimeric Proteins Class Communication Complex Crystallography Cyanobacterium Data Eubacterium Eukaryota Eukaryotic Cell Event Family Feasibility Studies G-Protein-Coupled Receptors GTP-Binding Proteins Genetic Techniques Goals Halobacterium salinarium Helix (Snails)Integral Membrane Protein Isotope Labeling Laboratories Light Mediating Membrane Membrane Proteins Methods Microbial Rhodopsins Microscopic Modeling Molecular Natronobacterium Neurospora crassa Peptides Phosphorylation Proteins Protons Raman Spectrum Analysis Research Resolution Retinal Retinal Pigments Rhodopsin Roentgen Rays Schiff Bases Sensory Sensory Rhodopsins Series Signal Transduction Signal Transduction Pathway Site Site-Directed Mutagenesis Spectroscopy, Fourier Transform Infrared Spectrum Analysis Structural Models Structure Techniques Testing Time Transducers Vertebral column Water Work absorption base chromophore deprotonation ear helix experience fungus in vivo mutant protonation receptor research study sensory rhodopsin I transmission process

项目摘要

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）：该项目的主要目的是了解一类信号转导的光受体功能的分子机制，称为感觉视紫红蛋白。其中包括来自古细菌的感觉视紫红素I和II（SRI和SRII），以及在真菌和真核生物中最近发现的感觉视淡蛋白的快速增长的列表。由于它们与细菌淡淡的蛋白质的密切关系，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，感觉视紫红蛋白为研究信号转导和膜蛋白相互作用提供了出色的模型系统。它们也与拟细菌中的趋化受体密切相关。感觉视opsins通过将信号传输到介导磷酸化级联反应的相关甲基换能器（HTR）来发挥作用。在初步研究中，已经从几个感觉视紫红蛋白及其突变体中获得了静态和时间分辨的FTIR差异光谱，这些光谱揭示了构象变化的存在，包括涉及Schiff碱基柜台和其他未识别残基的质子化变化的序列。我们已经证明了使用FTIR差异光谱法研究完整的感觉视紫红蛋白传染剂复合物的可行性，这些复合物是由融合蛋白的体内表达形成的。该数据揭示了由受体的相互作用和同源传感器的两个核心跨膜螺旋产生的信号。在拟议的研究中，将使用一系列基于红外的技术来检查正常和改良形式的感觉视紫红蛋白的光激发及其在微秒至几秒钟的时间尺度上发生的分子事件。这项研究将利用时间分辨，极化和ATR FTIR差异光谱以及FT-RAMAN光谱法。对感官视紫红蛋白的3D晶体的微观FTIR研究将提供有关X射线派生的X射线衍生结构的信息，这些结构可能会因晶格约束而改变。我们还将利用先前应用于细菌紫红素的先进遗传技术，该技术允许在特定部位将同位素标记和非本地氨基酸掺入感觉式视紫红蛋白及其传感器中。我们详细描述了一系列利用这些方法旨在测试最近提出的信号转导机制的一系列实验。