Habuchi Satoshi, Dedecker Peter, Hotta Jun-ichi, Flors Cristina, Ando Ryoko, Mizuno Hideaki, Miyawaki Atsushi, Hofkens Johan
Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium.
Photochem Photobiol Sci. 2006 Jun;5(6):567-76. doi: 10.1039/b516339k. Epub 2006 Mar 8.
Recently, reversible photoswitching in bulk samples or in individual molecules of Dronpa, a mutant of a green fluorescent protein (GFP)-like fluorescent protein, has been demonstrated. Intense irradiation at 488 nm changed Dronpa in a dim protonated form, and weak irradiation at 405 nm restored it to the bright deprotonated form. Here, we report on the mechanism of photoswitching of Dronpa by means of ensemble and single-molecule spectroscopy. Ensemble spectroscopy shows that the photoswitching can be described, in first approximation, by a three-state model including a deprotonated (B), a protonated (A1), and a photoswitched protonated (A2) forms of the chromophore. While the B and the A1 forms are in a ground state acid-base equilibrium, the B and the A2 forms are reversibly photoswitched upon irradiation with 488 and 405 nm light. At the single-molecule level, the on-times in fluorescence intensity trajectories excited at 488 nm decrease with increasing the excitation power, consistent with the photoswitching from the B to A2 form. The on-times agree well with expected values, which are calculated based on the ensemble spectroscopic properties of Dronpa. The fluorescence trajectory obtained with simultaneous dual-color excitation at 488 and 405 nm demonstrates reversible photoswitching between the B and the A2 forms at the single-molecule level. The efficiency of the photoswitching from the A2 to B form increased with increasing the excitation power of the 405 nm light. Our results demonstrate that Dronpa holds its outstanding photoswitching properties, based on a photo-induced protonation/deprotonation process, even at the single-molecule level.
最近，已证明在大量样品或类绿色荧光蛋白（GFP）荧光蛋白的突变体Dronpa的单个分子中存在可逆光开关现象。488nm的强辐射使Dronpa转变为暗质子化形式，而405nm的弱辐射将其恢复为亮去质子化形式。在此，我们通过系综光谱和单分子光谱报告Dronpa的光开关机制。系综光谱表明，光开关现象在一阶近似下可用一个三态模型来描述，该模型包括发色团的去质子化（B）、质子化（A1）和光开关质子化（A2）形式。虽然B和A1形式处于基态酸碱平衡，但B和A2形式在488nm和405nm光照射下可发生可逆光开关。在单分子水平上，488nm激发的荧光强度轨迹中的开启时间随激发功率增加而减小，这与从B形式到A2形式的光开关现象一致。开启时间与基于Dronpa系综光谱性质计算出的预期值非常吻合。在488nm和405nm同时双色激发下获得的荧光轨迹证明了在单分子水平上B和A2形式之间的可逆光开关。从A2形式到B形式的光开关效率随405nm光激发功率的增加而提高。我们的结果表明，即使在单分子水平上，Dronpa基于光诱导质子化/去质子化过程仍具有出色的光开关特性。
