Stoner-Ma Deborah, Jaye Andrew A, Ronayne Kate L, Nappa Jérôme, Meech Stephen R, Tonge Peter J
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
J Am Chem Soc. 2008 Jan 30;130(4):1227-35. doi: 10.1021/ja0754507. Epub 2008 Jan 8.
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 (< 1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is < or = 2.4 A. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins.
野生型绿色荧光蛋白（wtGFP）发色团的中性形式在激发时会发生激发态质子转移（ESPT），产生特征性的绿色（508nm）荧光。这种ESPT反应涉及一个质子从发色团的酚羟基传递到E222的离子化侧链，并导致在为中性物种优化的蛋白质环境（I态）中形成阴离子发色团。如在S65T和E222Q GFP突变体中发生的那样，E222的重新定向或替换会使ESPT反应失效，并导致中性发色团激发后绿色发射的丧失。此前已表明，在S65T GFP中引入第二个突变（H148D）可恢复绿色发射，这意味着ESPT再次成为可能。在E222Q/H148D突变体中也观察到了类似的绿色荧光恢复，表明D148是两个双突变体中ESPT反应的质子受体。通过使用稳态和超快时间分辨荧光及振动光谱，对S65T/H148D和E222Q/H148D中中性发色团激发后的荧光发射机制进行了探索。这些数据与单突变体S65T GFP的数据形成对比。时间分辨荧光研究表明，双突变体中I的形成非常迅速（<1ps），随后在皮秒时间尺度上进行振动冷却。时间分辨红外差谱与wtGFP或其阴离子突变体的谱明显不同。特别是，在皮秒红外差谱中没有明显的光谱特征表明D148的电离状态发生了改变，这导致有人提出在发色团的酚羟基和D148的侧链之间存在一个低势垒氢键（LBHB），基态和激发态具有不同的势能面。该模型与最近的高分辨率结构数据一致，其中供体和受体氧原子之间的距离小于或等于2.4埃。重要的是，这些研究表明wtGFP中的氢键网络可以被一个单一残基取代，这一观察结果如果得到充分探索，将有助于我们理解蛋白质内质子转移反应的各种要求。
