An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: Rewiring GFP
An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: Rewiring GFP
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DOI：
10.1021/ja0754507
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发表时间：
2008-01-30
影响因子：
15
通讯作者：
Tonge, Peter J.
中科院分区：
文献类型：
--
作者：
Stoner-Ma, Deborah;Jaye, Andrew A.;Tonge, Peter J.
野生型绿色荧光蛋白（wtGFP）中发色团的中性形式在激发时经历激发态质子转移（ESPT），导致特征性绿色（508 nm）荧光。该ESPT反应涉及从发色团的酚羟基到E222的离子化侧链的质子传递，并导致在针对中性物质（I* 状态）优化的蛋白质环境中形成阴离子发色团。如在S65 T和E222 Q GFP突变体中发生的，E222的重定向或替换使ESPT反应失效，并导致中性发色团激发后绿色发射的损失。先前，已经显示，将第二突变（H148 D）引入S65 T GFP允许恢复绿色发射，这意味着ESPT再次是可能的。对于E222 Q/H148 D突变体也观察到类似的绿色荧光恢复，表明D148是两种双突变体中ESPT反应的质子受体。通过稳态和超快时间分辨荧光光谱和振动光谱研究了S65 T/H148 D和E222 Q/H148 D中中性发色团激发后的荧光发射机制。将数据与单突变体S65 T GFP的数据进行对比。时间分辨荧光研究表明非常快速（
The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (