Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing the rates and pathways of ground-state proton transfer. We have utilized a specific isotopic labeling strategy in combination with one-dimensional 13C nuclear magnetic resonance (NMR) spectroscopy to install and monitor a 13C directly adjacent to the GFP chromophore ionization site. The chemical shift of this probe is highly sensitive to the protonation state of the chromophore, and the resulting spectra reflect the thermodynamics and kinetics of the proton transfer in the NMR line shapes. This information is complemented by time-resolved NMR, fluorescence correlation spectroscopy, and steady-state absorbance and fluorescence measurements to provide a picture of chromophore ionization reactions spanning a wide time domain. Our findings indicate that proton transfer in GFP is described well by a two-site model in which the chromophore is energetically coupled to a secondary site,likely the terminal proton acceptor of ESPT, Glu222. Additionally,experiments on a selection of GFP circular permutants suggest an importantrole played by the structural dynamics of the seventh β-strandin gating proton transfer from bulk solution to the buried chromophore.
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