2HGD
Structure of S65A Y66F GFP variant with an oxidized chromophore
2HGD の概要
エントリーDOI | 10.2210/pdb2hgd/pdb |
関連するPDBエントリー | 2HGY 2hcg |
分子名称 | Green fluorescent protein (2 entities in total) |
機能のキーワード | post-translational modification, cyclization, oxidation, fluorophore, gfp, 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 | 26825.14 |
構造登録者 | Barondeau, D.P.,Kassmann, C.J.,Tainer, J.A.,Getzoff, E.D. (登録日: 2006-06-26, 公開日: 2007-03-27, 最終更新日: 2023-11-15) |
主引用文献 | 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 Cited by 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: 17326633DOI: 10.1021/ja063983u 主引用文献が同じPDBエントリー |
実験手法 | X-RAY DIFFRACTION (1.6 Å) |
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