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首页> 外文期刊>Biochemistry >Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer
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Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer

机译：绿色荧光蛋白变体S65T / H148D中的超快激发态动力学。 3.激发态质子转移的短期和长期动力学

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Steady-state emission, femtosecond pump-probe spectroscopy, and time-correlated single-photon counting (TCSPC) measurements were used to study the photophysics and the excited-state proton transfer (ESPT) reactions in the green fluorescent protein (GFP) variant S65T/H148D at three pH values: 6.0, 7.9, and 9.5. Selective mutation of GFP caused a dramatic change in the steady-state and excited-state behavior as compared to the wild-type GFP (wt-GFP) studied earlier. An excitation wavelength dependence of the quantum yield of the strong emission band at 5 10 nm (I* band) indicates the competition between adiabatic and non-adiabatic excited-state proton-transfer reactions. Pump-probe measurements show that the signal buildup probed at 5 10 nm is biphasic, where 0. 8 of the signal amplitude is ultrashort, < 150 fs, and 0.2 of the signal decreases with a similar to 10 ps time constant. This effect is a summary result of adiabatic ESPT to the carboxylate group of Asp148 and nonradiative processes. When compared with the luminescence of wt-GFP, the steady-state intensity at 450 nm is lower by a factor of about 10. This very weak emission at 450 nm has a nonexponential decay. It is relatively pH insensitive and, at about 25 ps, is almost twice as long as in wt-GFP. The former exhibits a surprisingly small kinetic deuterium isotope effect (KDIE), compared with the KDIE of about 5 for wt-GFP. Such weak proton dependence may indicate that this emission comes from the species not directly involved in the ESPT. In contrast, pH and H/D isotope: dependence of the intense nonexponential luminescence decay of the S65T/HI48D deprotonated form measured at 510 nm may result from an isomerization-induced deactivation that is accompanied by the proton recombination quenching. The data are complementary, to the femtosecond time-resolved emission data obtained by ultrafast fluorescence up-conversion spectroscopy, found in the preceding paper (Shi et al.). The spectroscopic results are discussed on the basis of the detailed X-ray structure of the mutant published in the preceding paper (Shu et al.).
机译：稳态发射，飞秒泵浦探针光谱和与时间相关的单光子计数（TCSPC）测量用于研究绿色荧光蛋白（GFP）变体S65T的光物理和激发态质子转移（ESPT）反应/ H148D在三个pH值：6.0、7.9和9.5。与先前研究的野生型GFP（wt-GFP）相比，GFP的选择性突变导致稳态和激发态行为发生了巨大变化。激发波长在5 10 nm（I *波段）处的强发射带的量子产率的依赖性表明，绝热和非绝热激发态质子转移反应之间存在竞争。泵浦探针测量表明，在5 10 nm处探测到的信号是双相的，其中0. 8的信号幅度超短，<150 fs，0.2的信号以近似10 ps的时间常数减小。该效果是绝热ESPT对Asp148羧酸酯基团和非辐射过程的总结结果。与wt-GFP的发光相比，在450 nm处的稳态强度降低了约10倍。这种在450 nm处非常弱的发射具有非指数衰减。它对pH值不敏感，大约25 ps，几乎是wt-GFP的两倍。与wt-GFP的KDIE约为5相比，前者表现出惊人的动力学氘同位素效应（KDIE）。如此弱的质子依赖性可能表明这种排放来自与ESPT不直接相关的物种。相比之下，pH和H / D同位素：在510 nm处测量的S65T / HI48D去质子化形式的强烈非指数发光衰减的依赖性可能是由异构化诱导的失活伴随着质子重组猝灭而引起的。该数据与先前论文（Shi等人）中发现的通过超快速荧光上转换光谱法获得的飞秒时间分辨发射数据互补。根据在先前论文中发表的突变体的详细X射线结构（Shu等人），讨论了光谱结果。

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