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Color tuning of A first submission merged.pdf (3.35 MB)

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

submitted on 10.03.2020 and posted on 11.03.2020 by Chi-Yun Lin, Steven Boxer
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 preferable 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 the color of the neutral chromophore to be much more sensitive to protein electrostatics. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model compared with that of the anionic state, which requires only two forms. Specifically, an underlying zwitterionic charge transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to the protonated chromophore’s photoacidity and excited-state proton transfer (ESPT), 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 two-form model for donor–acceptor systems with localized electronic distributions.


National Institutes of Health GM118044

National Science Foundation CHE-1740645


Email Address of Submitting Author


Stanford University


United States

ORCID For Submitting Author


Declaration of Conflict of Interest