Rational Design of the Beta-Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing

Here, we report how the mutagenesis of the beta-bulge residues (D137 and W138) in mNeonGreen, a bright fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the beta-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching. Given the significance of sulfate in metabolic processes, we are now poised to apply SulfOFF-1 in living cells. We anticipate that fundamental insights gained from MD simulations can be integrated with protein engineering to design and expand this unique anion sensing function in the GFP family.