PROBING MEMBRANE PROTEIN DYNAMICS WITH ENERGY TRANSFER

跨膜离子转运是许多生理过程的基本方面， 流程. 为了理解分子水平上的传输现象， 活性物质的动态构象变化的结构信息 需要转运蛋白。 理想情况下，我们可以测定蛋白质 与离子中的单个基本步骤相关的结构变化 运输反应。 目前几乎没有获得这种信息的技术。 荧光能量转移技术已在我们的开发 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 转移已被用于初步研究，以定量衡量变化 在视网膜的细菌视紫红质的位置， 质子泵光循环 可以测量荧光能量转移 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地切断光化 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 作为受体的光循环中间体用以下物质测量 相敏检测 供体荧光和受体吸光度 可以分析振幅以确定最接近的距离， 光循环中吸收受体物质的脂质供体 中间体 因此，关于结构的高度具体的信息 得到了质子泵过程中反应中间体的分布。 的 建议的研究是将我们以前的工作扩展到更详细的研究， 细菌视紫红质（bR）和盐视紫红质（hR）的新研究， 视紫红质（sR）。 在bR工作中，将使用各种荧光供体。 用于定位视网膜在两个M中间状态的位置。 初步研究表明，视网膜和 在两个M州的位置。 位于不同位置的荧光供体 将研究脂质双层中的深度。 平行研究将 这提供了与bR的有用比较， 建立bR结果的一般性。 因为最大吸光度 hR和sR的光循环中间体中有90%得到了很好的分离， 蛋白质为这种能量转移的应用提供了更好的系统 法 对这些蛋白质的实验应该允许可视化 光循环中两个连续步骤的结构变化。 因此， 技术应该提供独特的结构信息的动态 膜结合蛋白质。

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.