Shedding light on the dark and weakly fluorescent states of green fluorescent proteins
Shedding light on the dark and weakly fluorescent states of green fluorescent proteins
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DOI：
10.1073/pnas.96.11.6177
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发表时间：
1999-05-25
影响因子：
11.1
通讯作者：
Langhoff, PW
中科院分区：
文献类型：
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作者：
Weber, W;Helms, V;Langhoff, PW
最近在单分子水平和溶液中对各种类似的绿色荧光蛋白(GFP)突变体的实验提供了以前未知的短寿命和长寿命的“暗”态以及相关的激发态衰变通道的证据。在这里，我们给出了中性、阴离子和两性离子GFP生色团基态和第一单重态激发态顺反光异构化路径的量子化学计算，从共同的角度解释了观察到的行为。结果表明，沿着这些异构化的不同质子态可能存在有利的无辐射衰变通道，这些异构化显然是通过锥形相交进行的。建议使用这些通道来解释所观察到的溶液中荧光急剧减少的现象。观察到的单分子快速闪烁归因于荧光阴离子和暗两性离子之间的转换，而缓慢的转换归因于阴离子和中性离子之间的转换。预测的非绝热交叉被认为在共同的基础上使各种实验观测的起源合理化，并可能对GFP中的光生物物理机制产生广泛的影响。
Recent experiments on various similar green fluorescent protein (GFP) mutants at the single-molecule level and in solution provide evidence of previously unknown short- and long-lived "dark" states and of related excited-state decay channels. Here, we present quantum chemical calculations on cis-trans photoisomerization paths of neutral, anionic, and zwitterionic GFP chromophores in their ground and first singlet excited states that explain the observed behaviors from a common perspective. The results suggest that favorable radiationless decay channels can exist for the different protonation states along these isomerizations, which apparently proceed via conical intersections. These channels are suggested to rationalize the observed dramatic reduction of fluorescence in solution. The observed single-molecule fast blinking is attributed to conversions between the fluorescent anionic and the dark zwitterionic forms whereas slow switching is attributed to conversions between the anionic and the neutral forms. The predicted nonadiabatic crossings are seen to rationalize the origins of a variety of experimental observations on a common basis and may have broad implications for photobiophysical mechanisms in GFP.