Determination of chromophore charge states in the low pH color transition of the fluorescent protein Rtms5H146S via time-dependent DFT

The red fluorescent protein Rtms5H146S displays a transition from blue (absorbance λmax 590 nm) to yellow (absorbance λmax ∼453 nm) upon titration to low pH. The pKa of the reaction depends on the concentration of halide, offering promise for new expressible halide sensors. The protonation state involved in the low pH form of the chromophore remains, however, ambiguous. We report calculated excitation energies of different protonation states of an RFP chromophore model. These suggest that the relevant titration site is the phenoxy moiety of the chromophore, and the relevant base and conjugate acid are anionic and neutral chromophore species, respectively.