FTIR Study of Bacteriorhodopsin

9419059 Rothschild该项目的中心目标是阐明细菌视紫红质（bR）中光驱动质子运输的分子机制。 在早期的工作中，它被证明，傅里叶变换红外（FTIR）差光谱可以提供详细的信息质子化状态，局部环境和特定的氨基酸残基的结构活性在bR光循环过程中的方向。 然而，限制未来进展的一个关键问题是将FTIR谱带分配给蛋白质中的各个基团。 虽然定点诱变（SDM）已成功地应用于特殊情况下，为此目的，它的使用是有限的，由于扰动诱导的蛋白质的结构和功能，如最近的研究所示的突变体Y185 F。此外，SDM不允许将谱带分配给特定肽基团的振动，这是研究蛋白质骨架构象变化的重要下一步，例如已经在bR的M和N中间体之间检测到的。 基于定点同位素标记（SDIL），现已发展出一种通用的谱带归属方法。 bR的SDIL类似物是通过使用特殊的氨酰化抑制tRNA的无细胞合成产生的。 这些SDIL-bR类似物具有与天然bR相同的性质，并且适用于FTIR-差示光谱。 在这种方法的第一个应用中，谱带被分配给特定酪氨酸侧链的振动以及bR骨架结构中的各个基团。 本研究将利用SDIL-FTIR技术系统地研究特定残基在bR质子泵中的作用。 我们的实验将由早期的研究提供信息的特定残基进行质子化的变化，可能参与一个假设的质子线，采取行动，以耦合的视网膜生色团的蛋白质，并参与蛋白质的二级结构变化。 最近发展起来的时间分辨傅里叶变换红外光谱差光谱法，偏振傅里叶变换红外光谱法和衰减全反射傅里叶变换红外光谱法也将用于这项研究。 在与J. Spudich实验室的合作中，SDIL-FTIR方法将扩展到感觉视紫红质I（sRI），盐生盐细菌中的趋光性光受体。 这个项目的主要目标是了解细菌视紫红质如何作为光驱动的质子泵发挥作用。 细菌视紫红质作为其他膜蛋白的原型，在细胞中进行基本过程，包括离子转运和能量转导。 FTIR-差分光谱是研究膜蛋白功能的一种有前途的新方法。 当与生物化学和遗传学方法相结合时，它有能力提供有关质子化状态，局部环境甚至蛋白质中特定基团方向的信息。 与固态NMR不同，红外光谱还可以探测蛋白质中的快速动态构象变化。 在本项目中，FTIR-差示光谱将结合三种方法：i）特定氨基酸的均匀同位素标记; ii）定点诱变（SDM）和定点同位素标记（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功能的详细图片，并为其他膜蛋白如何工作提供线索。 ***

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. ***