Ground-State Proton Transfer in Green Fluorescent Protein Measured by NMR

Abstract

Proton transfer plays an important role in the optical properties of fluorescent proteins. Excited-state proton transfer (ESPT) is responsible for wtGFP's anomalously large Stokes shift and a number of FPs have found use as in vivo pH indicators. Unlike ESPT, ground-state proton transfer cannot be synchronously initiated (i.e. with an excitation pulse) and must be studied more indirectly. We report an NMR method in which GFP is labeled with a 13C at the position adjacent the phenol hydroxyl on the chromophore. This position is a highly specific and sensitive reporter of the ionization state as measured by direct-detect 13C-NMR. Through lineshape analysis and time-resolved NMR spectroscopy we obtain kinetic parameters for proton transfer. We find that GFPs having internal proton transfer and capable of ESPT show rapid interconversion of the protonated and deprotonated states (on the order of microseconds) while GFPs which must transfer protons externally to the solvent have much slower equilibration (on the order of milliseconds). Parallel measurements with less direct fluorescence correlation spectroscopy reveal interesting discrepancies perhaps suggestive of light-driven structural dynamics.