Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets.
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.
Biomolecular NMR Spectroscopy, Leibniz Institute for Age Research, Fritz Lipmann Institute, Beutenbergstr. 11, 07745, Jena, Germany. pbell@fli-leibniz.de
Peter
P
Bellstedt
Thomas
T
Seiboth
Sabine
S
Häfner
Henriette
H
Kutscha
Ramadurai
R
Ramachandran
Matthias
M
Görlach
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure*
C438188:APTX protein, human; D000596:Amino Acids; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D009587:Nitrogen Isotopes; D009687:Nuclear Proteins