Photoinduced electron transfer in fluorescent proteins (FP) from the green fluorescent protein (GFP) family is a novel phenomenon. Depending on the task at hand, it is regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. Photooxidation in presence of an external oxidant commonly results in green-to-red photoconversion called oxidative redding. The mechanism of this process as well as the structural information of the red-form are still unknown. Overall process happens in timescale of seconds-to-minutes. We discovered that yellow FPs do not undergo redding, however, it is restored upon halide binding. We postulated that photoinduced electron transfer from the chromophore initiates the process followed by slow chemical steps resulting in the final red-form of the chromophore. To elucidate the mechanism of ET, we performed detailed first-principle calculations of the free energies for various ET pathways, as well as respective non-adiabatic couplings and Marcus rates. Calculations suggested that photoinduced ET proceeds through a hopping mechanism via Tyr145. In YFPs, the π-stacking of the chromophore with Tyr203 reduces its electron donating ability towards Tyr145, which can be restored by halide binding. The mechanism of the halide action is that it upsets the π-stacking with Tyr203. Point mutations confirmed that Tyr145 is a key residue controlling ET. Mutation of Tyr145 by less efficient electron acceptors resulted in highly photostable mutants. This strategy - calculation and disruption of ET pathways - may represent a new approach towards enhancing photostability of FPs.

Exploring photo-induced electron transfer leading to "oxidative redding" in fluorescent proteins

251st National Meeting (2016)

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