Tang Longteng, Wang Yanli, Liu Weimin, Zhao Yongxin, Campbell Robert E, Fang Chong
Department of Chemistry, Oregon State University , Corvallis, Oregon 97331, United States.
Department of Chemistry, University of Alberta , Edmonton, Alberta T6G 2G2, Canada.
J Phys Chem B. 2017 Apr 13;121(14):3016-3023. doi: 10.1021/acs.jpcb.7b01269. Epub 2017 Mar 30.
Fluorescent protein (FP)-based biosensors have become an important and promising tool to track metal ion movement inside living systems. Their working principles after light irradiation, however, remain elusive. To facilitate the rational design of biosensors, we dissect the fluorescence modulation mechanism of a newly developed excitation ratiometric green FP-based Ca biosensor, GEX-GECO1, using femtosecond stimulated Raman spectroscopy (FSRS) in the electronic excited state. Upon 400 nm photoexcitation, characteristic vibrational marker bands at ∼1180 and 1300 cm show concomitant decay and rise dynamics, probing the progression of an ultrafast excited state proton transfer (ESPT) reaction. The Ca-bound biosensor exhibits two distinct populations that undergo ESPT with ∼6 and 80 ps time constants, in contrast to one dominant population with a 25 ps time constant in the Ca-free biosensor. This result is supported by key structural constraints from molecular dynamics simulations with and without Ca. The blueshift of the ∼1265 cm C-O stretch mode unravels the vibrational cooling dynamics of the protonated chromophore regardless of Ca binding events. This unique line of inquiry reveals the essential structural dynamics basis of fluorescence modulation inside an excitation ratiometric protein biosensor, correlating the uncovered chromophore structural heterogeneity with different H-bonding configurations and intrinsic proton transfer rate in the photoexcited state.
基于荧光蛋白（FP）的生物传感器已成为追踪生物系统中金属离子移动的一种重要且有前景的工具。然而，光照射后它们的工作原理仍不清楚。为了便于生物传感器的合理设计，我们使用飞秒受激拉曼光谱（FSRS）在电子激发态下剖析了一种新开发的基于激发比率绿色荧光蛋白的钙生物传感器GEX-GECO1的荧光调制机制。在400nm光激发后，约1180和1300cm处的特征振动标记带显示出伴随的衰减和上升动力学，探测超快激发态质子转移（ESPT）反应的进程。与无钙生物传感器中一个具有25ps时间常数的主要群体相比，结合钙的生物传感器表现出两个不同的群体，它们经历ESPT的时间常数约为6和80ps。有无钙的分子动力学模拟得到的关键结构限制支持了这一结果。约1265cm处C-O伸缩模式的蓝移揭示了质子化发色团的振动冷却动力学，而与钙结合事件无关。这条独特的研究路线揭示了激发比率蛋白生物传感器内荧光调制的基本结构动力学基础，将发现的发色团结构异质性与光激发态下不同的氢键构型和固有质子转移速率联系起来。
