Ultrafast timescale structural fluctuations around a cofactor result in fluctuations of electronic state energies which can profoundly influence chemical activity, <i>e.g.</i> in charge-transfer reactions. We have used spectrally-resolved transient grating and time-resolved fluorescence spectroscopies to probe the influence of the chromophore environment in three families of red fluorescent proteins (FPs), in which the relative importance of structural <i>vs.</i> dynamic factors in generating large Stokes-shifted emission is not well understood. We demonstrate that picosecond timescale hydrogen bonding dynamics are the key to producing a large Stokes shift. In one study we examined solvation dynamics of TagRFP675 and point mutants at positions whose side chains interact with the chromophore N-acylimine oxygen atom. Electronic relaxation timescales of tens and hundreds of picoseconds were measured for all variants. Classical molecular dynamics simulations of these FPs revealed that variants with the largest Stokes shifts exhibit picosecond timescale rearrangements involving two sidechains and water molecules participating in hydrogen bonding interactions with the acylimine. This behavior contrasts with the dynamics uncovered in a study of the red FP mPlum, which has a simpler hydrogen-bonding network interacting with the N-acylimine and therefore shows two-state switching between direct and water-mediated conformers. As a third case, MD simulations of the FPs eQFP650 and eqFP670 show that the key difference between them corresponds to the increased flexibility of the hydrogen-bonding network interacting with the acylimine oxygen in eqFP670. We propose a simple model that explains the solvation dynamics of these far-red FPs. Our results demonstrate that far-red FPs from all known lineages exhibit fast hydrogen bonding dynamics in the acylimine region of the chromophore which lead to far red-shifted emission. Flexible hydrogen-bonding interactions similar to those discussed here could play an important role in stabilizing the electronic states of cofactors and/or substrates involved in enzymatic catalysis.

Picosecond hydrogen bond dynamics in proteins examined with femtosecond spectroscopy and molecular dynamics simulations

251st National Meeting (2016)

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