2FZU: Reduced Enolate Chromophore Intermediate For Gfp Variant

Citation:
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.
PDB ID: 2FZUDownload
MMDB ID: 38359
PDB Deposition Date: 2006/2/10
Updated in MMDB: 2012/11
Experimental Method:
x-ray diffraction
Resolution: 1.25  Å
Source Organism:
Similar Structures:
Biological Unit for 2FZU: monomeric; determined by author
Molecular Components in 2FZU
Label Count Molecule
Protein (1 molecule)
1
Green Fluorescent Protein
Molecule annotation
Chemicals (2 molecules)
1
1
2
1
* Click molecule labels to explore molecular sequence information.

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