1YHG

Uncyclized precursor structure of S65G Y66S V68G GFP variant

1YHG の概要

• エントリーDOI: 10.2210/pdb1yhg/pdb
• 関連するPDBエントリー: 1YHH 1YHI
• 分子名称: green fluorescent protein (2 entities in total)
• 機能のキーワード: chromophore uncyclized dimer, luminescent protein
• 由来する生物種: Aequorea victoria
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 53385.89

構造登録者

Barondeau, D.P.,Kassmann, C.J.,Tainer, J.A.,Getzoff, E.D. (登録日: 2005-01-07, 公開日: 2005-02-15, 最終更新日: 2024-02-14)

主引用文献

Barondeau, D.P.,Kassmann, C.J.,Tainer, J.A.,Getzoff, E.D.
Understanding GFP Chromophore Biosynthesis: Controlling Backbone Cyclization and Modifying Post-translational Chemistry.
Biochemistry, 44:1960-1970, 2005

PubMed Abstract: The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.

PubMed: 15697221
DOI: 10.1021/bi0479205

実験手法

X-RAY DIFFRACTION (2.5 Å)