title: Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C') and 3J(H beta,C') coupling constants in desulfovibrio vulgaris flavodoxin.
abstract: A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings 3J(HN,C') and 3J(H beta,C') in uniformly 13C-enriched proteins. The protocols for measuring accurate coupling constants are based on 1H,13C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95-105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of phi and chi 1 dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant 13C,15N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 3J(HN,C') coupling constants from a single spectrum.
authors.affiliation: Institut für Biophysikalische Chemie, Johann Wolfgang Goethe Universität Frankfurt am Main, Biozentrum, Germany.
authors.forename: J M
authors.initials: JM
authors.lastname: Schmidt
authors.forename: F
authors.initials: F
authors.lastname: Löhr
authors.forename: H
authors.initials: H
authors.lastname: Rüterjans
mesh_terms: D000595:Amino Acid Sequence; D003198:Computer Simulation; D016969:Desulfovibrio vulgaris / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016011:Normal Distribution; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012996:Solutions
chemical_list: D005418:Flavodoxin; D011994:Recombinant Proteins; D012996:Solutions