Excited-state dynamics in the enhanced green fluorescent protein mutant probed by picosecond time-resolved single photon counting spectroscopy

被引:86
|
作者
Cotlet, M
Hofkens, J [1 ]
Maus, M
Gensch, T
Van der Auweraer, M
Michiels, J
Dirix, G
Van Guyse, M
Vanderleyden, J
Visser, AJWG
De Schryver, FC
机构
[1] Katholieke Univ Leuven, Dept Chem, Celestijnenlaan 200F, B-3001 Heverlee, Belgium
[2] Katholieke Univ Leuven, Dept Agr, B-3001 Heverlee, Belgium
[3] Wageningen Univ Agr, Dept Biomol Sci, NL-6703 HA Wageningen, Netherlands
来源
JOURNAL OF PHYSICAL CHEMISTRY B | 2001年 / 105卷 / 21期
关键词
D O I
10.1021/jp003813i
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
The complex photophysics of the wild-type green fluorescent protein (GFP), one of the most popular fluorescent probes in biology, has been extensively documented in literature. The excited-state dynamics of GFP was explained by means of a model implying excited-state proton transfer (ESPT) and three forms of the chromophore, a protonated A form absorbing at 400 nm and two deprotonated I and B forms absorbing at around 475 nm, We report here a systematic picosecond time-resolved fluorescence study of the enhanced green fluorescent protein (EGFP) variant, carrying the Ser65-Thr and Phe64-Leu mutations. By means of multiple excitation wavelength time-resolved experiments, we were able to distinguish between the fluorescence decay times of the deprotonated I* and B* states (3.4 and 2.7 ns). Spectrally, we found the I form being red shifted in comparison with the B form, both in absorption and in emission. Evidence for an excited-state reaction, namely, proton transfer, is also reported. An additional protonated species is proposed in the photophysical scheme in order to explain the excited-state dynamics of EGFP on the basis of our results as well as previous reported data. Two alternative models are presented, both of them applicable also to the data reported in relation with wild-type GFP.
引用
收藏
页码:4999 / 5006
页数:8
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