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Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C′) and 3J(Hβ,C′) coupling constants in Desulfovibrio vulgaris flavodoxin

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Summary

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(Hβ,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 ϕ and X 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′) and 100 3J(Hβ,C′) coupling constants are obtained from a single spectrum.

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Abbreviations

ANOVA:

analysis of variances

COSY:

correlated spectroscopy

E.COSY:

exclusive correlation spectroscopy

FMN:

flavin mononucleotide

HMQC:

heteronuclear multiple-quantum coherence

HSQC:

heteronuclear single-quantum coherence

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Authors and Affiliations

  1. Institut für Biophysikalische Chemie, Johann Wolfgang Goethe Universität Frankfurt am Main, Biozentrum, Marie Curie Strasse 9, D-60439, Frankfurt am Main, Germany

    Jürgen M. Schmidt, Frank Löhr & Heinz Rüterjans

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  1. Jürgen M. Schmidt
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  2. Frank Löhr
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  3. Heinz Rüterjans
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Schmidt, J.M., Löhr, F. & Rüterjans, H. Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C′) and 3J(Hβ,C′) coupling constants in Desulfovibrio vulgaris flavodoxin. J Biomol NMR 7, 142–152 (1996). https://doi.org/10.1007/BF00203824

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