2hgd
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Structure of S65A Y66F GFP variant with an oxidized chromophore
OverviewOverview
The green fluorescent protein (GFP) creates its fluorophore by promoting, spontaneous peptide backbone cyclization and amino acid oxidation, chemistry on its own Ser65, Tyr66, Gly67 tripeptide sequence. Here we use, high-resolution crystallography and mutational analyses to characterize, GFP variants that undergo backbone cyclization followed by either, anticipated chromophore synthesis via Y66F Calpha-Cbeta double-bond, formation or unprecedented loss of a Y66F benzyl moiety via Calpha-Cbeta, bond cleavage. We discovered a Y66F cleavage variant that subsequently, incorporates an oxygen atom, likely from molecular oxygen, at the Y66, Calpha position. The post-translational products identified from these, Y66F GFP structures support a common intermediate that partitions between, Calpha-Cbeta oxidation and homolytic cleavage pathways. Our data indicate, that Glu222 is the branchpoint control for this partitioning step and also, influences subsequent oxygen incorporation reactions. From these results, we propose mechanisms for Y66F Calpha-Cbeta cleavage, oxygen, incorporation, and chromophore biosynthesis with shared features that, include radical chemistry. By revealing how GFP and RFP protein, environments steer chemistry to favor fluorophore biosynthesis and, disfavor alternative reactivity, we identify strategies for protein, design. The proposed, common, one-electron oxidized, radical intermediate, for post-translation modifications in the GFP family has general, implications for how proteins drive and control spontaneous, post-translational chemical modifications in the absence of metal ions.
About this StructureAbout this Structure
2HGD is a Single protein structure of sequence from Aequorea victoria. Full crystallographic information is available from OCA.
ReferenceReference
The Case of the Missing Ring: Radical Cleavage of a Carbon-Carbon Bond and Implications for GFP Chromophore Biosynthesis., Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED, J Am Chem Soc. 2007 Mar 21;129(11):3118-26. Epub 2007 Feb 28. PMID:17326633
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