2FWQ
Reduced enolate chromophore intermediate for Y66H GFP variant
Summary for 2FWQ
| Entry DOI | 10.2210/pdb2fwq/pdb |
| Descriptor | Green fluorescent protein, MAGNESIUM ION (3 entities in total) |
| Functional Keywords | beta barrel, chromophore, biosynthesis, intermediate, enolate, dithionite, luminescent protein |
| Biological source | Aequorea victoria |
| Total number of polymer chains | 1 |
| Total formula weight | 26753.32 |
| Authors | Barondeau, D.P.,Tainer, J.A.,Getzoff, E.D. (deposition date: 2006-02-02, release date: 2006-03-14, Last modification date: 2024-10-30) |
| Primary citation | Barondeau, D.P.,Tainer, J.A.,Getzoff, E.D. Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis. J.Am.Chem.Soc., 128:3166-3168, 2006 Cited by PubMed Abstract: 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. PubMed: 16522096DOI: 10.1021/ja0552693 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.4 Å) |
Structure validation
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