PROBING MEMBRANE PROTEIN DYNAMICS WITH ENERGY TRANSFER

通过能量转移探测膜蛋白动力学

• 批准号: 3290086
• 负责人: GREGORY T DEWEY
• 金额: $ 5.6万
• 依托单位: UNIVERSITY OF DENVER (COLORADO SEMINARY)
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290086
• 关键词: Halobacteriaceae; bacteriorhodopsins; cell membrane; fluorescence spectrometry; fluorescent dye /probe; gel filtration chromatography; ion transport; membrane lipids; photochemistry

Transmembrane ionic transport is a fundamental aspect of many physiological processes. To understand transport phenomena at a molecular level, structural information on the dynamic, conformational changes of active transport proteins is required. Ideally, one would determine protein structural changes associated with individual, elementary steps in the ion transport reaction. Few techniques exist for obtaining such information. A fluorescence energy transfer technique has been developed in our laboratory that provides specific structural information on membrane-protein dynamics. Phase Modulation of Fluorescence Energy Transfer has been used in initial studies to quantitatively measure changes in the location of the retinal in bacteriorhodopsin during its proton-pumping photocycle. Fluorescence energy transfer can be measured from lipid donors to photocycle intermediates. The population of the photocycle intermediate is modulated by mechanically chopping the actinic light driving bacteriorhodopsin's photosystem. The resulting quenching of fluorescence due to energy transfer from the lipid donor to a specific photocycle intermediate acting as an acceptor is measured with phase-sensitive detection. The donor fluorescence and acceptor absorbance amplitudes can be analyzed to determine the distance of closest approach of the lipid donor to the absorbing acceptor species in the photocycle intermediate. Thus, highly specific information concerning the structure of reaction intermediates in the proton pumping process is obtained. The proposed study is to extend our previous work to a more detailed study of bacteriorhodopsin (bR) and to new studies on halorhodopsin (hR) and sensory rhodopsin(sR). In the bR work, a variety of fluorescent donors will be used to locate the position of retinal in the two M intermediate states. Preliminary studies show a significant difference between the retinal location in the two M states. Fluorescent donors located at different depths in the lipid bilayer will be investigated. Parallel studies will be initiated on hR and sR. This provides a useful comparison to bR and will establish the generality of the bR results. Because the absorbance maxima of the photocycle intermediates of hR and sR are well separated, these proteins offer a better system for the application of this energy transfer technique. Experiments on these proteins should allow a visualization of structural changes at two consecutive steps in the photocycle. Thus, this technique should provide unique structural information on the dynamics of membrane-bound proteins.

跨膜离子转运是许多生理学的一个基本方面 流程。 为了理解分子水平上的传输现象， 有关活性物质动态、构象变化的结构信息 需要转运蛋白。 理想情况下，人们可以确定蛋白质 与离子中的单个基本步骤相关的结构变化 运输反应。 获取此类信息的技术很少。 我们已开发出荧光能量转移技术 提供特定结构信息的实验室 膜蛋白动力学。 荧光能量的相位调制 初始研究中已使用转移来定量测量变化 细菌视紫红质在其视网膜中的位置 质子泵浦光循环。 可测量荧光能量转移 从脂质供体到光循环中间体。 的人口 光循环中间体通过机械切割光化剂来调节 光驱动细菌视紫红质的光系统。 由此产生的淬火 由于能量从脂质供体转移到特定的物质而产生的荧光 作为受体的光循环中间体的测量 相敏检测。 供体荧光和受体吸光度 可以分析振幅以确定最近接近的距离 光循环中吸收受体物种的脂质供体 中间的。 因此，有关结构的高度具体的信息 获得质子泵浦过程中的反应中间体。 这 拟议的研究是将我们之前的工作扩展到更详细的研究 细菌视紫红质 (bR) 以及盐视紫红质 (hR) 和感觉的新研究 视紫红质(SR)。 在 bR 工作中，多种荧光供体将被 用于定位视网膜在两个M个中间状态下的位置。 初步研究显示视网膜之间存在显着差异 位于两个M州。 荧光供体位于不同的位置 将研究脂质双层的深度。 平行研究将 在 hR 和 sR 上启动。 这提供了与 bR 和 will 的有用比较 建立 bR 结果的普遍性。 因为吸光度最大值 hR 和 sR 的光循环中间体被很好地分离，这些 蛋白质为这种能量转移的应用提供了更好的系统 技术。 对这些蛋白质的实验应该可以直观地看到 光循环中两个连续步骤的结构变化。 因此，这个 技术应提供有关动力学的独特结构信息 膜结合蛋白。