We recently developed the red fluorescent protein (RFP) mCherry-XL, which is threefold brighter than its predecessor, mCherry, with a directed evolution approach using fluorescence-lifetime flow cytometry selections. The enhanced brightness is due to a significant decrease in the non-radiative decay rate underlying its increase in fluorescence quantum yield. To examine the dynamic role of the four mutations that distinguish the two RFPs and closely-related variants, we employed microsecond-timescale, all-atom molecular dynamics simulations to sample their ground state conformational landscapes. The simulations revealed the significance of the I197R mutation, which leads to the formation of multiple hydrogen-bonded contacts. The triad of interactions observed between residues K70, E148 and 197R is also seen in mScarlet, another RFP of unrelated origin, but of comparably high brightness. These substitutions in mCherry-XL increased the rigidity of the β-barrel, for example as shown by increased hydrogen-bonding in the chromophore region and decreased root-mean-square backbone deviations. Furthermore, mCherry-XL showed significantly less ns-timescale breathing of the gap between β-7 and β-10 strands. This gap was previously shown to be the most flexible region of mCherry, permitting entry of O2 into the barrel. We also characterize the role played by the sidechain of residue 161 using a combination of MD simulations and in-vitro experimental characterization. We find this position is critical to the steric interactions that perturb the chromophore electronic structure. MD simulations also help us to recognize a network of hydrogen-bonded interactions between the chromophore, the residue 143, 163 and 59, which can potentially impact the electron distribution of the chromophore. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution.
Supplementary Information (Sections S1 to S11) have been provided.