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

Abstract

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.

PEG-mCherry interactions beyond classical macromolecular crowding

“…Mukherjee et al also observed two states of the gap, albeit with slightly different populations than those observed here. 61 This difference in state populations may be due to differences in force field choice, simulation system, or conformational sampling (i.e., 2 µs vs. 20 µs in the present work).…”

“…This gap is a vestige of the missing tetrameric interactions disrupted by mutations introduced to design a monomeric FP. 85 Previous studies have shown that the β7-β10 gap undergoes structural transitions between closed and open states, 61 allowing molecular oxygen to diffuse into the barrel and affect the photostability of the chromophore. 55,85 To determine the width of the β7-β10 gap, we measured the distance between three residues on β7 (E144, A145, S146) and three residues on β10 (I197, K198, L199).…”

“…The chemical and physical characteristics of the chromophore's environment are, in turn, affected by the structure and dynamics of the protein, as well as by the solvent environment. [60][61][62] Recent experimental work has shown that the fluorescence lifetime of various FPs can decrease in the presence of elevated concentrations of crowders. 51 τ was observed to monotonically decrease with increasing crowder concentrations above 30% fractional volume occupancy (FVO) with no corresponding spectral changes.…”

Influence of fluorescence lifetime selections and conformational flexibility on brightness of FusionRed variants

“…We performed all-atom molecular dynamics simulations, spanning approximately 500 ns, 31 to evaluate the influence of single substitutions at positions 175, 42, 159, and 218 in FR. (SI Section S2b-e).…”

Influence of Fluorescence Lifetime Selections and Conformational Flexibility on Brightness of FusionRed Variants

“…MD Simulations. We performed all-atom molecular dynamics simulations, spanning 500 ns, 31 to evaluate the influence of single substitutions at positions 175, 42, 159, and 218 in FR (Supporting Information Section S2). Computational details are provided in Supporting Information Section S1e.…”