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首页> 外文期刊>Biochemistry >Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties
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Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties

机译：绿色荧光蛋白变体S65T / H148D中的超快激发态动力学。 2.异常的光物理性质

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In the preceding accompanying paper [Shu, X., et al. (2007) Biochemistry 46, 12005-12013], the 1.5 angstrom resolution crystal structure of green fluorescent protein (GFP) variant S65T/H148D is presented, and the possible consequences of an unusual short hydrogen bond (<= 2.4 angstrom) between the carboxyl oxygen of Asp148 and the phenol oxygen of the chromophore are discussed. This work reports the femtosecond time-resolved emission of this variant at pH 5.6 by ultrafast fluorescence upconversion spectroscopy. Following excitation at 400 nm, green fluorescence is observed at 510 nm with a rise on a time scale that is faster than the 170 fs instrument response. Time-resolved emission spectra at 140 K also exhibit the immediate appearance of green fluorescence, and this extremely fast process is hardly affected by deuteration of exchangeable protons. These results appear to be dramatically different from those of wild-type GFP, in which the green fluorescence at 508 nm is produced on the picosecond time scale as a result of excited-state proton transfer from the state that is excited at 400 nm. The unique features observed in S65T/ H148D and apparent ultrafast excited-state proton transfer are discussed in light of evidence for multiple states underlying the band at around 415 nm, as suggested by steady-state fluorescence spectra. The behavior of these different states may explain the novel photophysical properties observed for this GFP variant, including the ultrafast green fluorescence and the absence of completely matched decay in blue fluorescence. It is speculated that two different orientations of the Asp introduced at position 148, not distinguishable by chromatography, mass spectrometry, or X-ray crystallography, give rise to the two functionally distinct populations.
机译：在前面的随附论文中[Shu，X.，et al。 （2007）Biochemistry 46，12005-12013]，提出了绿色荧光蛋白（GFP）变体S65T / H148D的1.5埃分辨率晶体结构，以及羧基之间异常的短氢键（<= 2.4埃）的可能后果讨论了Asp148的氧和发色团的酚氧。这项工作通过超快速荧光上转换光谱法报告了该变体在pH 5.6的飞秒时间分辨发射。在400 nm激发后，在510 nm处观察到绿色荧光，其时间尺度的上升快于170 fs仪器响应。 140 K的时间分辨发射光谱也立即显示出绿色荧光，并且这种极快的过程几乎不受可交换质子氘的影响。这些结果似乎与野生型GFP显着不同，在野生型GFP中，由于激发态质子从400 nm激发态转移而在皮秒级时产生了508 nm的绿色荧光。根据稳态荧光光谱显示的证据，讨论了在S65T / H148D中观察到的独特特征以及明显的超快激发态质子转移，这些证据表明在415 nm附近处于谱带下方的多个状态。这些不同状态的行为可能解释了此GFP变体观察到的新光物理性质，包括超快绿色荧光和蓝色荧光中不存在完全匹配的衰减。据推测，在位置148处引入的Asp的两个不同取向，无法通过色谱法，质谱法或X射线晶体学法来区分，从而产生了两个功能上不同的群体。

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