Viscosity-dependent fluorescence decay of the GFP chromophore in solution due to fast internal conversion
Viscosity-dependent fluorescence decay of the GFP chromophore in solution due to fast internal conversion
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DOI：
10.1021/jp014713v
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发表时间：
2002-08-01
影响因子：
3.3
通讯作者：
Michel-Beyerle, ME
中科院分区：
文献类型：
--
作者：
Kummer, AD;Kompa, C;Michel-Beyerle, ME
275 K 下的时间分辨荧光测量表明，绿色荧光蛋白 (GFP) 模型发色团的激发态寿命从乙醇等低粘度溶剂中的亚皮秒 (eta = 1.7 cP) 大幅增加到甘油中的 30 ps (eta = 9.9 x 10(3) cP)，并在 150 K 的甘油玻璃中达到 2.1 ns。激发态衰变和基态恢复动力学的研究表明内部转换是荧光寿命短的原因。它们的粘度依赖性反映了具有相当大振幅的运动，该运动被粘性阻力阻尼并且超过了热激活，正如在不同温度下的测量得出的结论。在溶液中，模型生色团的中性和阴离子形式类似地不发出荧光，而野生型 GFP 和突变体的去质子化形式几乎不进行内部转化。因此，该蛋白质选择性地限制特定质子化状态下生色团的运动自由度。
Time-resolved fluorescence measurements at 275 K show that the excited-state lifetime of a model chromophore of the green fluorescent protein (GFP) substantially increases from subpicoseconds in low-viscosity solvents such as ethanol (eta = 1.7 cP) to 30 ps in glycerol (eta = 9.9 x 10(3) cP) and reaches 2.1 ns in glycerol glass at 150 K. At high temperatures the similarity of excited-state decay and ground-state recovery kinetics indicates internal conversion being responsible for the short fluorescence lifetimes. Their viscosity dependence reflects on a motion with a considerable amplitude that is damped by viscous drag and outweighs thermal activation as is concluded from measurements at different temperatures. In solution the neutral and the anionic forms of the model chromophore are similarly nonfluorescent in contrast to wild-type GFP and mutants where the deprotonated form is hardly undergoing internal conversion. Thus, the protein selectively restricts motional degrees of freedom of the chromophore in specific protonation states.