FTIR Study of Bacteriorhodopsin

细菌视紫红质的 FTIR 研究

• 批准号: 9419059
• 负责人: Kenneth Rothschild
• 金额: $ 30万
• 依托单位: Trustees of Boston University
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9419059&HistoricalAwards=false
• 关键词: FTIR; Study; Bacteriorhodopsin

9419059 Rothschild The central goal of this project is the elucidation of the molecular mechanism of light-driven proton transport in bacteriorhodopsin (bR). In earlier work, it was demonstrated that Fourier transform infrared (FTIR) difference spectroscopy can provide detailed information about the protonation state, local environment and orientation of specific amino acid residues which are structurally active during the bR photocycle. However, a key problem limiting future progress is the assignment of FTIR bands to individual groups in a protein. While site-directed mutagenesis (SDM) has been successfully applied in special cases for this purpose, its use is limited due to perturbations induced in the structure and function of the protein as illustrated by recent studies on the mutant Y185F. In addition, SDM does not permit the assignment of bands to the vibrations of specific peptide groups, an essential next step for investigating protein backbone conformational changes such as have been detected between the M and N intermediates of bR. A general method of band assignment has now been developed based on site-directed isotope labeling (SDIL). SDIL analogs of bR are produced by cell-free synthesis using specially aminoacylated suppressor tRNAs. These SDIL-bR analogs have identical properties as native bR and are suitable for FTIR-difference spectroscopy. In the first application of this approach, bands have been assigned to the vibrations of specific tyrosine side-chains as well as to individual groups in the bR backbone structure. In this research, SDIL-FTIR will be used to investigate systematically the role of specific residues in bR proton pumping . Our experiments will be guided by earlier studies which have provided information about specific residues which undergo protonation changes, may participate in a hypothesized proton wire, act to couple the retinal chromophore to the protein and are involved in secondary structural changes of the protei n. Recently developed methods for time-resolved FTIR-difference spectroscopy, polarized FTIR and attenuated total reflection FTIR will also be used in this research. In collaboration with the J. Spudich laboratory, the SDIL-FTIR approach will be extended to sensory-rhodopsin I (sRI), a phototaxis light-receptor in Halobacteria salinarium. %%% The primary goal of this project is understanding how bacteriorhodopsin functions as a light-driven proton pump. Bacteriorhodopsin serves as a prototype for other membrane proteins which carry out essential process in cells including ion transport and energy transduction. A promising new approach for studying how membrane proteins function is FTIR-difference spectroscopy. When combined with biochemical and genetic methods it has the power to provide information about the protonation state, local environment and even orientation of specific groups in a protein. Unlike solid-state NMR, infrared spectroscopy can also probe rapid dynamic conformational changes in proteins. In this project, FTIR- difference spectroscopy will be combined with three methods: i) uniform isotope labeling of specific amino acids; ii) site-directed mutagenesis (SDM) and site-directed isotope labeling (SDIL) to investigate bacteriorhodopsin. If successful, these studies will provide a detailed picture of how bacteriorhodopsin functions and provide clues to how other membrane proteins work. ***

本项目的中心目标是阐明细菌视紫红质（bR）中光驱动质子传输的分子机制。在早期的研究中，傅里叶变换红外（FTIR）差分光谱可以提供bR光循环过程中具有结构活性的特定氨基酸残基的质子化状态、局部环境和取向的详细信息。然而，限制未来进展的一个关键问题是将FTIR波段分配给蛋白质中的单个基团。虽然定点突变（site-directed mutagenesis， SDM）已经成功地应用于特殊情况，但其应用受到限制，因为最近对突变体Y185F的研究表明，这种方法会引起蛋白质结构和功能的扰动。此外，SDM不允许将波段分配给特定肽基团的振动，这是研究蛋白质主链构象变化的必要步骤，例如在bR的M和N中间体之间已经检测到。目前，基于位点定向同位素标记（SDIL）已经发展出一种通用的波段分配方法。bR的SDIL类似物是利用特殊的氨基酰化抑制因子trna通过无细胞合成产生的。这些sdi -bR类似物具有与天然bR相同的性质，适合于ftir差分光谱。在这种方法的第一次应用中，波段被分配给特定酪氨酸侧链的振动以及bR主链结构中的单个基团。在本研究中，sdi - ftir将用于系统地研究特定残基在bR质子泵送中的作用。我们的实验将在早期研究的指导下进行，这些研究提供了特定残基的信息，这些残基经历质子化变化，可能参与假设的质子线，将视网膜发色团与蛋白质偶联，并参与蛋白质的二级结构变化。最近开发的时间分辨FTIR差分光谱，偏振FTIR和衰减全反射FTIR也将用于本研究。与J. Spudich实验室合作，sdl - ftir方法将扩展到感觉视紫红质I (sRI)，盐杆菌中的一种趋光性光受体。这个项目的主要目标是了解细菌视紫红质如何作为一个光驱动质子泵起作用。细菌视紫红质是其他膜蛋白的原型，这些膜蛋白在细胞中进行重要的过程，包括离子运输和能量转导。ftir差分光谱是研究膜蛋白功能的一种很有前途的新方法。当与生物化学和遗传方法相结合时，它有能力提供有关蛋白质中特定基团的质子化状态、局部环境甚至取向的信息。与固态核磁共振不同，红外光谱还可以探测蛋白质的快速动态构象变化。在本项目中，FTIR-差分光谱将与三种方法相结合：1)特定氨基酸的均匀同位素标记；ii)位点定向诱变（SDM）和位点定向同位素标记（SDIL）来研究细菌视紫红质。如果成功，这些研究将提供细菌视紫红质如何发挥作用的详细图片，并为其他膜蛋白如何工作提供线索。＊＊＊