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The neutral or A state of the green fluorescent protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the protein environment and accounts for the large Stokes shift of GFP in response to near UV excitation. Its color tuning mechanism has been largely overlooked, as it is less preferred for imaging applications than the redder anionic or B state. Past studies, based on site-directed mutagenesis or solvatochromism of the isolated chromophore, have concluded that its color tuning range is much narrower than its anionic counterpart. However, as we performed extensive investigation on more GFP mutants, we found that the color of the neutral chromophore can be more sensitive to protein electrostatics than can the anionic counterpart. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model as compared to that of the anionic state, which requires only two forms ( J. Am. Chem. Soc. 2019, 141, 15250-15265). Specifically, an underlying zwitterionic charge-transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to excited-state proton transfer (ESPT) of the protonated chromophore, we infer design principles of the GFP chromophore as a photoacid through the color tuning mechanisms of both protonation states. The three-form model could also be applied to similar biological and nonbiological dyes and complements the failure of the two-form model for donor-acceptor systems with localized ground-state electronic distributions.

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore