Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis

Nadya V. Pletneva, Vladimir Z. Pletnev, Konstantin A. Lukyanov, Nadya G. Gurskaya, Ekaterina A. Goryacheva, Vladimir I. Martynov, Alexander Wlodawer, Zbigniew Dauter, Sergei Pletnev

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)


The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence (λex = 480 nm, λem = 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with Cα and C β atoms in the sp3 hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation (λ = 250-300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 Cα=C β bond and the conjugated system of π-electrons. Structure-based site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.

Original languageEnglish
Pages (from-to)15978-15984
Number of pages7
JournalJournal of Biological Chemistry
Issue number21
Publication statusPublished - 21 May 2010
Externally publishedYes


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