2HGD

Structure of S65A Y66F GFP variant with an oxidized chromophore

Summary for 2HGD

• Entry DOI: 10.2210/pdb2hgd/pdb
• Related: 2HGY 2hcg
• Descriptor: Green fluorescent protein (2 entities in total)
• Functional Keywords: post-translational modification, cyclization, oxidation, fluorophore, gfp, luminescent protein
• Biological source: Aequorea victoria
• Total number of polymer chains: 1
• Total formula weight: 26825.14

Authors

Barondeau, D.P.,Kassmann, C.J.,Tainer, J.A.,Getzoff, E.D. (deposition date: 2006-06-26, release date: 2007-03-27, Last modification date: 2023-11-15)

Primary citation

Barondeau, D.P.,Kassmann, C.J.,Tainer, J.A.,Getzoff, E.D.
The Case of the Missing Ring: Radical Cleavage of a Carbon-Carbon Bond and Implications for GFP Chromophore Biosynthesis
J.Am.Chem.Soc., 129:3118-3126, 2007

PubMed Abstract: 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.

PubMed: 17326633
DOI: 10.1021/ja063983u

Experimental method

X-RAY DIFFRACTION (1.6 Å)