title: A simplified recipe for assigning amide NMR signals using combinatorial 14N amino acid inverse-labeling.
abstract: Assignment of backbone amide proton resonances is one of the most time-consuming stages of any protein NMR study when the protein samples behave non-ideally. A robust and convenient NMR procedure for analyzing spectra of marginal-to-low quality is helpful for high-throughput structure determination. The 14N selective- and inverse-labeling method is a candidate solution. Here, we present a simplified protocol for assigning protein backbone amide NMR signals. When 14N inversely labeled residues are present in a protein, their backbone NH cross peaks vanish from the protein's 1H-(15)N HSQC spectrum, and thus, their chemical shifts can be readily identified by a process of elimination. Some metabolically related amino acids, for example, Ile, Leu, and Val, cannot be individually incorporated but can be inversely labeled together. We optimized and simplified the protocol and M9-based medium formula for the 14N selective- and inverse-labeling method without any additives. Our approach should be cost-effective, because the method could be additively applied stepwise, even when the proteins of interest were found to be non-ideal.
authors.affiliation: Division of Structural Biology, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo, Kobe, 650-0017, Japan. hiroakih@med.kobe-u.ac.jp
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Hiroaki
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Umetsu
authors.forename: Yo-ichi
authors.initials: Y
authors.lastname: Nabeshima
authors.forename: Minako
authors.initials: M
authors.lastname: Hoshi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kohda
mesh_terms: D000577:Amides; D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry*; D016229:Amyloid beta-Peptides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D053583:Interleukin-1beta / Q000737:chemistry; D007553:Isotope Labeling; D009584:Nitrogen; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D011506:Proteins / Q000737:chemistry*
chemical_list: D000577:Amides; D000596:Amino Acids; D016229:Amyloid beta-Peptides; D053583:Interleukin-1beta; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011506:Proteins; C098994:amyloid beta-protein (1-43); D009584:Nitrogen