Excited State Proton Transfer in the Red Fluorescent Protein mKeima

Abstract

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 lifetime ~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.

Monomeric red fluorescent proteins with a large Stokes shift

“…It is expected that in the ground state, the pKa of the D167/E167 carboxylates is lower than the pKa of the Y66 side chain hydroxyl, suggesting that the chromophores can be stabilized in their neutral forms. This is consistent with the excitation spectra of both LSS-mKates having maxima at ∼460 nm, which is characteristic for the neutral DsRed-like chromophore (23)(24)(25). Upon excitation of the neutral chromophore, the pKa values drop by several units (23) leading to the formation of an anionic chromophore, which subsequently emits a red photon.…”

“…Upon excitation of the neutral chromophore, the pKa values drop by several units (23) leading to the formation of an anionic chromophore, which subsequently emits a red photon. Therefore, we propose that the D167/E167 residues are mainly responsible for the large Stokes shifts in LSS-mKates by enabling an excited-state proton transfer (ESPT) from the chromophore tyrosine hydroxyl to the side chain of the 167 residue, as occurs in mKeima (24,25). Notably, mKate mutants with the residue 167 substituted with Lys (a proton acceptor amino acid) found during our screening also had a large Stokes shift.…”

Blue protein with red fluorescence

“…In bacterial phytochromes, it has been proposed that proton transfer and hydrogen bond interactions play a significant role in determining their fluorescence quantum yield (28). Excited-state proton transfer (ESPT) in GFP (40)(41)(42)(43) and its variant proteins has been known for decades, which is critical in the observed redshift in their fluorescence emission (12,14,44).…”

Rot fluoreszierende Proteine: spezielle Anwendungen in der Bildgebung und Perspektiven

“…Ein rationales Design neuer RFPs beruht auf Kenntnissen über die Prinzipien von Bildung und Funktion der Chromophore. Studien zu Kristallographie, [2f,g, 96] Mutagenese, Isotopenuntersuchungen, [2i, 97] Fließgleich-gewicht-und zeitauflçsender Spektroskopie, [97,98] Massenspektrometrie, [37, 96a] elektronischen Strukturberechnungen [99] und der Synthese von Modellchromophoren [100] wurden durchgeführt, um die Umwandlungen der roten Chromophore aufzuklären. Es gibt inzwischen genügend Informationen, um neue RFPs auf der Grundlage einer rationalen Strategie zu entwerfen.…”