2fwq

From Proteopedia

Reduced enolate chromophore intermediate for Y66H GFP variant

PDB ID 2fwq

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Structural highlights

2fwq is a 1 chain structure with sequence from Aequorea victoria. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.4Å
Ligands:	,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

GFP_AEQVI Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca(2+)-activated photoprotein aequorin.

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

Publication Abstract from PubMed

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.

Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis.,Barondeau DP, Tainer JA, Getzoff ED J Am Chem Soc. 2006 Mar 15;128(10):3166-8. PMID:16522096^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Barondeau DP, Tainer JA, Getzoff ED. Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis. J Am Chem Soc. 2006 Mar 15;128(10):3166-8. PMID:16522096 doi:10.1021/ja0552693