RAMAN STUDIES OF LIGHT TRANSDUCTION IN HALOBACTERIA

这项研究的长期目标是确定分子机制 光->能量和光->信息在细菌中的传递 视紫红质 尽管跨膜生物技术的产生和利用 质子浓度梯度是生物能量学中的一个基本要素，我们 不知道任何离子泵的结构或机制。 细菌视 (BR)是在盐生盐杆菌中发现的内在膜蛋白， 作为一个光驱动的质子泵的功能，和盐视紫红质（HR）是一个光- 驱动氯离子泵。 这两种蛋白质都含有一种视网膜蛋白。 进行13-反式-> 13-顺式光异构化的辅基 驱动离子运输的细胞 共振拉曼振动光谱将 用于研究发色团和蛋白质结构的变化， BR和HR的光循环，以确定其分子机制。 这项工作 应该有助于我们了解其他离子泵的机制， 化学动相驱动的酶 感觉视紫红质（SR）是一种类似的 在盐细菌中发现的一种含有视黄酸的蛋白质， 趋光受体 可见共振拉曼光谱将用于 确定母体颜料中发色团的结构及其 中间体的 这些SR结果与 视觉视紫红质的光感和BR和HR的离子泵应 有助于定义这些内在膜的基本功能概念 proteins. 这项工作也将是重要的，在发展我们的一般 受体结构和功能的知识。 具体建议 实验是： (1)时间分辨，可见共振拉曼振动光谱将是 使用双色泵浦-探测器从BR和HR中间体获得 pH、盐和温度的函数。 动力学 将模拟振幅与时间的关系以确定光循环方案。 (2)时间分辨紫外共振拉曼光谱将获得的 蛋白质的不同氨基酸侧链和酰胺组分， 定义了光循环中的蛋白质结构变化。 BR生长在 同位素氨基酸和BR位点特异性突变体将用于分配 并解释这些数据。 (3)时间分辨可见拉曼实验将在BR现场进行 特定的突变体，以确定点突变如何干扰发色团 结构和相互作用。 这将有助于确定位置， 在不同的发色团中的取向和相互作用 中间体的 (4)K、L、M、N和O中间体的可见拉曼振动光谱 将详细分析，以更彻底地定义 发色团和发色团-蛋白质相互作用。 视网膜同位素 导数将被用来定义这些分配， 场计算。 (5)SR-I和SR-II颜料的共振拉曼光谱及其 将获得中间体并用于测定发色团 每个物种的结构与同位素视网膜衍生物。

The long term goal of this research is to determine the molecular mechanism of light -> energy and light -> information transduction in bacterial rhodopsins. Although the generation and utilization of a trans-membrane proton concentration gradient is a fundamental element in bioenergetics, we do not know the structure or mechanism of any ion pumps. Bacteriorhodopsin (BR) is an intrinsic membrane protein found in Halobacterium halobium that functions as a light-driven proton pump, and halorhodopsin (HR) is a light- driven chloride ion pump. Both of these proteins contain a retinal prosthetic group which undergoes a 13-trans ->13-cis photoisomerization that drives ion transport. Resonance Raman vibrational spectroscopy will be used to study the chromophore and protein structural changes in the photocycles of BR and HR to determine their molecular mechanism. This work should help us to understand the mechanism of other ion pumps and of chemiosomotically-driven enzymes. Sensory rhodopsin (SR) is a similar retinal-containing protein found in halobacteria that functions as a phototactic receptor. Visible resonance Raman spectroscopy will be used to determine the structure of the chromophore in the parent pigment(s) and its intermediates. The comparison of these SR results with the mechanism of light-sensing in visual rhodopsin and of ion pumping in BR and HR should help to define the basic functional concepts of these intrinsic membrane proteins. This work will also be important in developing our general knowledge of receptor structure and function. Specific proposed experiments are: (1) Time-resolved, visible resonance Raman vibrational spectra will be obtained of the BR and HR intermediates using a two-color, pump-probe configuration as a function of pH, salt and temperature. The kinetic amplitudes vs. time will be modeled to determine the photocycle scheme. (2) Time-resolved UV resonance Raman spectra will be obtained of the different amino acid side chain and amide components of the protein to define the protein structural changes in the photocycle. BR grown on isotopic amino acids and BR site specific mutants will be used to assign and interpret these data. (3) Time-resolved visible Raman experiments will be performed on BR site specific mutants to determine how point mutations perturb the chromophore structure and interactions. This will help to define the location, orientation and interactions of the chromophore in the various intermediates. (4) Visible Raman vibrational spectra of the K, L, M, N and O intermediates will be analyzed in detail to more thoroughly define the structure of the chromophore and chromophore-protein interactions. Retinal isotopic derivatives will be used to define these assignments and to refine force field calculations. (5) Resonance Raman spectra of the SR-I and SR-II pigments and their intermediates will be obtained and used to determine the chromophore structure in each species with isotopic retinal derivatives.