Alternative E.COSY techniques for the measurement of 3J(C (i) (') (-1),C (i) (beta) ) and (3) J(H (i) (N) ,C (i)
1Institut für Biophysikalische Chemie, Johann Wolfgang Goethe-Universität Frankfurt am Main, Biozentrum N230, 1. OG, Marie Curie-Strasse 9, D-60439, Frankfurt, Germany.
Abstract:
Experiments are proposed for the measurement of the vicinal coupling constants between beta-carbons and either amide protons of the same or carbonyl carbons of the preceding amino acid residue in 13C/15N-labeled proteins. Both couplings depend on the backbone torsional angle phi. The three-dimensional pulse sequences give rise to E.COSY-like multiplet patterns in which heteronuclear one-bond couplings separate the doublet components corresponding to the two spin states of the respective passive nuclei. Thus, in contrast to previously published pulse schemes which employed the homonuclear 1J(Calpha,Cbeta) interaction, difficulties due to overlap of spectral regions of active and passive spins are avoided. A major drawback of the novel sequences is their limited sensitivity. Nevertheless, application to Desulfovibrio vulgaris flavodoxin yielded coupling constants for more than 85% of all non-glycine and non-proline residues.
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