Schenk Andreas, Ivanchenko Sergey, Röcker Carlheinz, Wiedenmann Jörg, Nienhaus G Ulrich
Department of Biophysics, University of Ulm, Ulm, Germany.
Biophys J. 2004 Jan;86(1 Pt 1):384-94. doi: 10.1016/S0006-3495(04)74114-4.
Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.
红色荧光蛋白是基于荧光的生命科学研究中的重要工具。最近，我们引入了eqFP611，一种从四色海葵中获得的具有优异特性的红色荧光蛋白。在这里，我们通过对蛋白质溶液使用荧光相关光谱法，研究了eqFP611以及作为对照的drFP583（DsRed）在亮态和暗态之间亚毫秒级的光驱动分子内动力学。一个具有一个暗态和两个荧光态的三态模型描述了两种蛋白质在不同激发波长下闪烁动力学的功率依赖性。它涉及两个光驱动的构象转变。我们还研究了固定在表面的单个（单体）eqFP611分子的光动力学。与聚乙二醇覆盖的玻璃表面结合的eqFP611的闪烁速率和暗态分数与在溶液中测量的结果相同，表明结合的荧光蛋白表现相同。在10毫秒时间尺度上观察到了第二个慢得多的闪烁过程。eqFP611分子沉积在裸玻璃表面上产生明亮的荧光，没有任何可检测到的闪烁，并且光漂白产率降低了10倍以上。这些观察结果强调了荧光蛋白中蛋白质运动与光物理过程之间的紧密联系。
