Abstract
Constant-time 3D heteronuclear relayed E.COSY [Schmidt et al. (1996) J. Biomol. NMR, 7, 142–152], as based on generic 2D small-flip-angle HMQC-COSY [Schmidt et al. (1995) J. Biomol. NMR, 6, 95–105], has been modified to allow for quantitative determination of heteronuclear three-bond 3 J(Hα,Cγ) couplings. The method is applicable to amino acid spin topologies with carbons in the γ position which lack attached protons, i.e. to asparagine, aspartate, and aromatic residues in uniformly 13C-enriched proteins. The pulse sequence critically exploits heteronuclear triple-quantum coherence (HTQC) of CH2 moieties involving geminal Hβ proton pairs, taking advantage of improved multiple-quantum relaxation properties, at the same time avoiding scalar couplings between those spins involved in multiple-quantum coherence, thus yielding E.COSY-type multiplets with a splitting structure that is simpler than with the original scheme. Numerical least-squares 2D line-shape simulation is used to extract 3 J(Hα,Cγ) coupling constants which are of relevance to side-chain χ1 dihedral-angle conformations in polypeptides. Methods are demonstrated with recombinant 15N,13C-enriched ribonuclease T1 and Desulfovibrio vulgaris flavodoxin with bound oxidized FMN.
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Löhr, F., Pérez, C., Köhler, R. et al. Heteronuclear relayed E.COSY revisited: Determination of 3J(Hα,Cγ) couplings in Asx and aromatic residues in proteins. J Biomol NMR 18, 13–22 (2000). https://doi.org/10.1023/A:1008385202639
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DOI: https://doi.org/10.1023/A:1008385202639