J Biol Chem, 2010, 285(21):15978-15984

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

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 Cαand Cβatoms in the sp3hybridization. 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=Cβbond 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.

Pletneva NV, Pletnev VZ, Lukyanov KA, Gurskaya NG, Goryacheva EA, Martynov VI, Wlodawer A, Dauter Z, Pletnev S

IBCH: 4974
Ссылка на статью в журнале: http://www.jbc.org/lookup/doi/10.1074/jbc.M109.092320
Кол-во цитирований на 11.2023: 24
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