title: Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis.
abstract: 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 (lambda(ex) = 480 nm, lambda(em) = 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 C(alpha) and C(beta) atoms in the sp(3) 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 (lambda = 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 C(alpha)=C(beta) bond and the conjugated system of pi-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.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 GSP, Moscow V-437, Russia. pletnevs@mail.nih.gov
authors.forename: Nadya V
authors.initials: NV
authors.lastname: Pletneva
authors.forename: Vladimir Z
authors.initials: VZ
authors.lastname: Pletnev
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Lukyanov
authors.forename: Nadya G
authors.initials: NG
authors.lastname: Gurskaya
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Goryacheva
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Martynov
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Dauter
authors.forename: Sergei
authors.initials: S
authors.lastname: Pletnev
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D037402:Hydrozoa / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D010084:Oxidation-Reduction / Q000528:radiation effects; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
chemical_list: D049452:Green Fluorescent Proteins