Theoretical Computer‐Aided Mutagenic Study on the Triple Green Fluorescent Protein Mutant S65T/H148D/Y145F

Green fluorescent protein (GFP) mutant S65T/H148D has been proposed to host a photocycle that involves an excited‐state proton transfer between the chromophore (Cro) and the Asp148 residue and takes place in less than 50 fs without a measurable kinetic isotope effect. It has been suggested that the interaction between the unsuspected Tyr145 residue and the chromophore is needed for the ultrafast sub‐50 fs rise in fluorescence. To verify this, we have performed a computer‐aided mutagenic study to introduce the additional mutation Y145F, which eliminates this interaction. By means of QM/MM molecular dynamics simulations and time‐dependent density functional theory studies, we have assessed the importance of the Cro–Tyr145 interaction and the solvation of Asp148 and shown that in the triple mutant S65T/H148D/Y145F a significant loss in the ultrafast rise of the Stokes‐shifted fluorescence should be expected.