2FWQ

Reduced enolate chromophore intermediate for Y66H GFP variant

2FWQ の概要

• エントリーDOI: 10.2210/pdb2fwq/pdb
• 分子名称: Green fluorescent protein, MAGNESIUM ION (3 entities in total)
• 機能のキーワード: beta barrel, chromophore, biosynthesis, intermediate, enolate, dithionite, luminescent protein
• 由来する生物種: Aequorea victoria
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 26753.32

構造登録者

Barondeau, D.P.,Tainer, J.A.,Getzoff, E.D. (登録日: 2006-02-02, 公開日: 2006-03-14, 最終更新日: 2024-10-30)

主引用文献

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

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: 16522096
DOI: 10.1021/ja0552693

実験手法

X-RAY DIFFRACTION (1.4 Å)