RAMAN STUDIES OF LIGHT TRANSDUCTION IN HALOBACTERIA

这项研究的长期目标是确定分子机制。 细菌中的光-能量和光-信息转导 视紫红质。虽然跨膜蛋白的产生和利用 质子浓度梯度是生物能量学的一个基本元素，我们 不知道任何离子泵的结构或机制。细菌视紫红质 (BR)是在盐生盐杆菌中发现的一种固有的膜蛋白，它 起到光驱动质子泵的作用，而卤视紫质(HR)是一种光- 驱动氯离子泵。这两种蛋白质都含有视网膜 经过13-反式-13-顺式光异构化的假体基团 这推动了离子的运输。共振拉曼振动光谱学将 用来研究生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。这部作品 应该有助于我们理解其他离子泵的机制，以及 化学小体驱动的酶。感觉性视紫红质(SR)是一种类似的 在嗜盐细菌中发现的含有视网膜的蛋白质，其功能是 趋光感受器。可见共振拉曼光谱将用于 亲本色素(S)及其发色团结构的测定 中间体。这些随机共振的结果与机理的比较。 视觉视紫红质中的光感觉以及BR和HR中的离子泵应 有助于定义这些固有膜的基本功能概念 蛋白质。这项工作对发展我们的将军也将是重要的 了解受体的结构和功能。具体建议 实验包括： (1)时间分辨的可见共振拉曼振动光谱将是 用双色泵浦探头获得BR和HR中间体 作为pH、盐和温度的函数的构型。动能 将对振幅与时间的关系进行建模，以确定光循环方案。 (2)将获得时间分辨的紫外共振拉曼光谱 不同氨基酸侧链和酰胺组分的蛋白质 定义光周期中蛋白质结构的变化。BR生长在 同位素氨基酸和BR位点特定突变体将被用来分配 并对这些数据进行解读。 (3)将在BR位上进行时间分辨的可见光拉曼实验 确定点突变如何扰乱发色团的特定突变体 结构和相互作用。这将有助于定义位置， 生色团在不同物种中的定位和相互作用 中间体。 (4)K、L、M、N和O中间体的可见拉曼振动光谱 将被详细分析以更彻底地定义 生色团和生色团-蛋白质的相互作用。视网膜同位素 将使用导数来定义这些分配并提炼作用力 现场计算。 (5)SR-I和SR-II颜料及其颜料的共振拉曼光谱 将获得中间体并用于测定发色团 每个物种的结构都有同位素视网膜衍生品。

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.