Tang Longteng, Liu Weimin, Wang Yanli, Zhao Yongxin, Oscar Breland G, Campbell Robert E, Fang Chong
Department of Chemistry, Oregon State University, Corvallis, Oregon 97331-4003 (USA).
Chemistry. 2015 Apr 20;21(17):6481-90. doi: 10.1002/chem.201500491. Epub 2015 Mar 11.
Imaging Ca(2+) dynamics in living systems holds great potential to advance neuroscience and cellular biology. G-GECO1.1 is an intensiometric fluorescent protein Ca(2+) biosensor with a Thr-Tyr-Gly chromophore. The protonated chromophore emits green upon photoexcitation via excited-state proton transfer (ESPT). Upon Ca(2+) binding, a significant population of the chromophores becomes deprotonated. It remains elusive how the chromophore structurally evolves prior to and during ESPT, and how it is affected by Ca(2+) . We use femtosecond stimulated Raman spectroscopy to dissect ESPT in both the Ca(2+) -free and bound states. The protein chromophores exhibit a sub-200 fs vibrational frequency shift due to coherent small-scale proton motions. After wavepackets move out of the Franck-Condon region, ESPT gets faster in the Ca(2+) -bound protein, indicative of the formation of a more hydrophilic environment. These results reveal the governing structure-function relationship of Ca(2+) -sensing protein biosensors.
对活体细胞内钙离子动态变化进行成像，对推动神经科学和细胞生物学的发展具有巨大潜力。G-GECO1.1是一种具有苏氨酸-酪氨酸-甘氨酸发色团的强度型荧光蛋白钙离子生物传感器。质子化发色团通过激发态质子转移（ESPT）在光激发时发出绿色荧光。钙离子结合后，大量发色团会发生去质子化。目前尚不清楚发色团在ESPT之前和过程中是如何进行结构演变的，以及它是如何受到钙离子影响的。我们使用飞秒受激拉曼光谱来剖析在无钙离子和有钙离子结合状态下的ESPT过程。由于相干的小规模质子运动，蛋白质发色团表现出低于200飞秒的振动频率偏移。波包移出弗兰克-康登区域后，在有钙离子结合的蛋白质中ESPT变得更快，这表明形成了一个更亲水的环境。这些结果揭示了钙离子传感蛋白生物传感器的结构-功能主导关系。
