1yhh
From Proteopedia
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Uncyclized precursor structure of S65A Y66S G67A GFP variant
Overview
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.
About this Structure
1YHH is a Single protein structure of sequence from Aequorea victoria. Full crystallographic information is available from OCA.
Reference
Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry., Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED, Biochemistry. 2005 Feb 15;44(6):1960-70. PMID:15697221
Page seeded by OCA on Wed Nov 21 06:46:44 2007
