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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.

SUBMITTER: Pletneva NV

PROVIDER: S-EPMC2871466 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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

Pletneva Nadya V NV Pletnev Vladimir Z VZ Lukyanov Konstantin A KA Gurskaya Nadya G NG Goryacheva Ekaterina A EA Martynov Vladimir I VI Wlodawer Alexander A Dauter Zbigniew Z Pletnev Sergei S

The Journal of biological chemistry 20100309 21

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 ...[more]

PMID: 20220148

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Project description:The chromophore of the green fluorescent protein (GFP) is critical for probing environmental influences on fluorescent protein behavior. Using the aqueous system as a bridge between the unconfined vacuum system and a constricting protein scaffold, we investigate the steric and electronic effects of the environment on the photodynamical behavior of the chromophore. Specifically, we apply ab initio multiple spawning to simulate five picoseconds of nonadiabatic dynamics after photoexcitation, resolving the excited-state pathways responsible for internal conversion in the aqueous chromophore. We identify an ultrafast pathway that proceeds through a short-lived (sub-picosecond) imidazolinone-twisted (I-twisted) species and a slower (several picoseconds) channel that proceeds through a long-lived phenolate-twisted (P-twisted) intermediate. The molecule navigates the non-equilibrium energy landscape via an aborted hula-twist-like motion toward the one-bond-flip dominated conical intersection seams, as opposed to following the pure one-bond-flip paths proposed by the excited-state equilibrium picture. We interpret our simulations in the context of time-resolved fluorescence experiments, which use short- and long-time components to describe the fluorescence decay of the aqueous GFP chromophore. Our results suggest that the longer time component is caused by an energetically uphill approach to the P-twisted intersection seam rather than an excited-state barrier to reach the twisted intramolecular charge-transfer species. Irrespective of the location of the nonadiabatic population events, the twisted intersection seams are inefficient at facilitating isomerization in aqueous solution. The disordered and homogeneous nature of the aqueous solvent environment facilitates non-selective stabilization with respect to I- and P-twisted species, offering an important foundation for understanding the consequences of selective stabilization in heterogeneous and rigid protein environments.

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

Publications

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

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