PROBING MEMBRANE PROTEIN DYNAMICS WITH ENERGY TRANSFER

跨膜离子转运是许多生理学研究的基本方面 流程。为了在分子水平上理解传输现象， 关于Active的动态、构象变化的结构信息 运输蛋白是必需的。理想情况下，人们会测定蛋白质 与离子中的单个基本步骤相关的结构变化 运输反应。几乎没有什么技术可以获得这样的信息。 我们开发了一种荧光能量转移技术。 提供特定结构信息的实验室 膜蛋白动力学。荧光能量的位相调制 转移在最初的研究中被用来定量测量变化 细菌视紫红质在视网膜中的位置 质子泵浦光循环。可以测量荧光能量传递 从脂质供体到光循环中间体。这个国家的人口 光循环中间体是通过机械地切割光化性物质来调制的 光驱动细菌视紫红质的光系统。由此产生的猝灭 从脂类供体到特定分子的能量转移产生的荧光 作为受体的光环中间体用来测量 相敏检测。供体荧光和受体吸光度 可以分析振幅来确定最接近的距离 光循环中吸收受体物种的脂类供体 中级的。因此，关于结构的高度具体的信息 得到了质子泵浦过程中反应中间产物的浓度。这个 建议的研究是将我们先前的工作扩展到更详细的研究 细菌视紫红质(BR)和卤视紫质(HR)与感觉的新研究 视紫红质(Sr)。在BR工作中，各种荧光供体将被 用于定位处于两个M中间状态的视网膜的位置。 初步研究表明，视网膜与 位于两个M状态的位置。位于不同地点的荧光供体 将对脂质双层的深度进行调查。平行研究将是 在HR和SR上启动。这提供了与BR和Will进行有用的比较 建立BR结果的一般性。因为吸光度最大值 的光循环中间体被很好地分离，这些 蛋白质为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允许可视化 光循环中连续两个阶段的结构变化。因此，这一点 技术应该提供关于动力学的独特的结构信息 膜结合蛋白。

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.