Conformational Dynamics of Rhodopsin and Bacteriorhodopsin

利用荧光偏振和能量转移研究了视紫红质和细菌视紫红质这两种光激活蛋白的构象动力学。多年来，这些蛋白质分别为受体作用和离子转运提供了重要的模型系统。本研究的目的是研究由于构象转变导致的蛋白质灵活性的变化。这些系统对于这一目的特别方便，因为很容易用光诱导转变。这些实验应该提供关于蛋白质动力学如何影响构象转变的独特信息。位相寿命光谱被用来测量细菌视紫红质在质子泵浦光循环过程中的荧光能量转移和荧光偏振的变化。这项新技术将允许监测生色团视网膜位置的变化，以寻找存在于毫秒级时间尺度上的中间体。在视紫红质的工作中，荧光标记被贴在位于羧基尾巴上的一个位置上，该位置被认为是将尾巴锚定在膜上。这个部位的膜位置将通过荧光能量转移来确定。荧光各向异性测量将被用来描述“系留尾巴”的运动。最后，将研究理论模型以提供分子动力学和构象变化之间的联系。

Fluorescence polarization and energy transfer is used to probe the conformational dynamics of two light-activated proteins, rhodopsin and bacteriorhodopsin. Over the years, these proteins have provided important model systems for receptor action and ion transport, respectively. The goal of the present study is to investigate changes in protein flexibility as a result of conformational transitions. These systems are especially convenient for this purpose because of the ease of inducing transitions with light. These experiments should provide unique information on how protein dynamics influence conformational transitions. Phase lifetime spectroscopy is used to measure changes in fluorescence energy transfer and fluorescence polarization in bacteriorhodopsin as it proceeds through its proton-pumping photocycle. This novel technique will allow changes in position of the chromophore, retinal, to be monitored for intermediates which exist on the millisecond time scale. In the rhodopsin work, fluorescence labels are attached to a site located on the carboxyl tail which is thought to anchor the tail to the membrane. The membrane location of the this site will be established using fluorescence energy transfer. Fluorescence anisotropy measurements will be used to characterize the motion of the "tethered tail". Finally, theoretical models will be investigated to provide link between molecular dynamics and conformational changes.