Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets.
Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets.
J Biomol NMR. 2013 Aug 14;
Authors: Bellstedt P, Seiboth T, Häfner S, Kutscha H, Ramachandran R, Görlach M
Abstract
NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23*kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ("scrambling") of NH and CO groups in a standard Escherichia coli expression host is provided.
PMID: 23943084 [PubMed - as supplied by publisher]
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