Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness.

The 3-fold higher brightness of the recently developed mCherry-XL red fluorescent protein (FP) compared to its progenitor, mCherry, is due to a significant decrease in the nonradiative decay rate underlying its increased fluorescence quantum yield. To examine the structural and dynamic role of the four mutations that distinguish the two FPs and closely related variants, we employed microsecond time scale, all-atom molecular dynamics simulations. The simulations revealed that the I197R mutation leads to the formation of multiple hydrogen-bonded contacts and increased rigidity of the β-barrel. In particular, mCherryXL showed reduced nanosecond time scale breathing of the gap between the β7 and β10-strands, which was previously shown to be the most flexible region of mCherry. Together with experimental results, the simulations also reveal steric interactions of residue 161 and a network of hydrogen-bonding interactions of the chromophore with residues at positions 59, 143, and 163 that are critical in perturbing the chromophore electronic structure. 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.