Excited State Proton Transfer in the Red Fluorescent Protein mKeima

Abstract

MKeima is an unusual monomeric red fluorescent protein (λ^em_max ∼620 nm) that is maximally excited in the blue (λ^ex_max ∼440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 Å resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the emission contains a green component (λ^em_max ∼535 nm), enhanced by deuterium substitution, presumably resulting from reduced proton transfer efficiency. Ultrafast pump/probe studies reveal a rising component in the 610 nm emission with a lifetime of ∼4 ps, characterizing the rate of proton transfer. Mutation of Asp157 to neutral Asn changes the chromophore resting charge state to anionic (λ^ex_max ∼565 nm, λ^em_max ∼620 nm). Thus, excited state proton transfer (ESPT) explains the large Stokes shift. This work unambiguously characterizes green emission from the protonated acylimine chromophore of red fluorescent proteins.