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    Structural Evidence for an Enolate Intermediate in GFP Fluorophore Biosynthesis
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    Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2006, 128, 10, 3166–3168
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    https://doi.org/10.1021/ja0552693
    Published February 16, 2006
    Copyright © 2006 American Chemical Society
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    Abstract

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    The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1/2 = 39 min-1) upon exposure to air. Isolation of this enolate intermediate will now allow specific probing of the rate-limiting oxidation step for fluorophore biosynthesis in GFP and its red fluorescent protein homologues. Such targeted characterizations may lead to the design of faster maturing proteins with enhanced applications in biotechnology and cell biology. Moreover, our results reveal how the GFP protein environment mimics enzyme systems, by stabilizing an otherwise high energy enolate intermediate to achieve its post-translational modification.

    Copyright © 2006 American Chemical Society

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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2006, 128, 10, 3166–3168
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ja0552693
    Published February 16, 2006
    Copyright © 2006 American Chemical Society
    Request reuse permissions

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