Excited state proton transfer in the red fluorescent protein mKeima

J. Nathan Henderson, Maire F. Osborn, Nayden Koon, Rinat Gepshtein, Dan Huppert, S. James Remington

Research output: Contribution to journalArticlepeer-review

Abstract

(Chemical Equation Presented) mKeima is an unusual monomeric red fluorescent protein (λemmax∼620 nm) that is maximally excited in the blue (λexmax ∼ 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 (λemmax ∼ 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 (λexmax ∼ 565 nm, λemmax ∼ 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.

Original languageEnglish
Pages (from-to)13212-13213
Number of pages2
JournalJournal of the American Chemical Society
Volume131
Issue number37
DOIs
StatePublished - 23 Sep 2009

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