Exploring the effects of mutagenesis on FusionRed using excited state QM/MM dynamics and classical force field simulations

Fluorescent proteins (FPs) are a powerful tool for examin- ing tissues, cells, and subcellular components in vivo and in vitro. FusionRed is a particular FP variant mutated from mKate2 that, in addition to lower cytotoxicity and aggregation rates, has shown potential for acting as a tunable photoswitch. This was posited to stem partially from the presence of a bulky side chain at position 158 and a further stabilizing residue at position 157. In this work, we apply computational techniques including classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics simulations (QM/MM) to explore the effect of mutagenesis at these locations in FusionRed on the chromophore structure, the excited state surface, and relative positional stability of the chromophore in the protein pocket. We find specific connections between the statistical sampling of the underlying protein structure and the nonradiative decay mechanisms from excited state dynamics. A single mutation (C158I) which restricts the motion of the chromophore via a favorable hydrophobic interaction corresponds to an increase in FQY, while a second rescue mutation (C158I-A157N) partially restores the flexibility of the chromophore and photoswitchability with favorable water interactions on the surface of the protein that counteracts the original interaction. We suggest that applying this understanding of structural features that inhibit or favor rotation on the excited state can be applied for rational design of new, tunable and red photoswitches.