An Alternate Proton Acceptor for Excited-State Proton Transfer in Green Fluorescent Protein: Rewiring GFP
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- 作者：Deborah Stoner-Ma ; Andrew A. Jaye ; Kate L. Ronayne ; Jé ; rô ; me Nappa ; Stephen R. Meech ; Peter J. Tonge
- 刊名：Journal of the American Chemical Society
- 出版年：2008
- 出版时间：January 30, 2008
- 年：2008
- 卷：130
- 期：4
- 页码：1227 - 1235
- 全文大小：374K
- 年卷期：v.130,no.4(January 30, 2008)
- ISSN：1520-5126
文摘
The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoesexcited-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 ionizedside chain of E222, and results in formation of the anionic chromophore in a protein environment optimizedfor the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T andE222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitationof 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 similarrecovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 isthe proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emissionfollowing excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been exploredthrough the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. Thedata are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicatevery rapid (<1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecondtime scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionicmutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra thatwould correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrierhydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain ofD148, with different potential energy surfaces for the ground and excited states. This model is consistentwith recent high-resolution structural data in which the distance between the donor and acceptor oxygenatoms is 
le.gif">2.4 Å. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can bereplaced by a single residue, an observation which, when fully explored, will add to our understanding ofthe various requirements for proton-transfer reactions within proteins.
le.gif">2.4 Å. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can bereplaced by a single residue, an observation which, when fully explored, will add to our understanding ofthe various requirements for proton-transfer reactions within proteins.