Acid-base catalysis and crystal structures of a least evolved ancestral GFP-like protein undergoing green-to-red photoconversion

Hanseong Kim, Timothy J. Grunkemeyer, Chintan Modi, Liqing Chen, Raimund Fromme, Mikhail V. Matz, Rebekka Wachter

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


In green-to-red photoconvertible fluorescent proteins, a three-ring chromophore is generated by the light-activated incorporation of a histidine residue into the conjugated π-system. We have determined the pH-rate profile and high- and low-pH X-ray structures of a least evolved ancestor (LEA) protein constructed in the laboratory based on statistical sequence analysis. LEA incorporates the minimal number of substitutions necessary and sufficient for facile color conversion and exhibits a maximal photoconversion quantum yield of 0.0015 at pH 6.1. The rate measurements provide a bell-shaped curve, indicating that the reaction is controlled by the two apparent pKa values, 4.5 ± 0.2 and 7.5 ± 0.2, flanking the chromophore pKa of 6.3 ± 0.1. These data demonstrate that the photoconversion rate of LEA is not proportional to the A-form of the GFP-like chromophore, as previously reported for Kaede-type proteins. We propose that the observed proton dissociation constants arise from the internal quadrupolar charge network consisting of Glu222, His203, Glu148, and Arg69. Increased active site flexibility may facilitate twisting of the chromophore upon photoexcitation, thereby disrupting the charge network and activating the Glu222 carboxylate for the abstraction of a proton from a carbon acid. Subsequently, the proton may be delivered to the Phe64 carbonyl by a hydrogen-bonded network involving Gln42 or by means of His65 side chain rotations promoted by protein breathing motions. A structural comparison of LEA with the nonphotoconvertible LEA-Q42A variant supports a role for Gln42 either in catalysis or in the coplanar preorganization of the green chromophore with the His65 imidazole ring.

Original languageEnglish (US)
Pages (from-to)8048-8059
Number of pages12
Issue number45
StatePublished - Nov 12 2013

ASJC Scopus subject areas

  • Biochemistry


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