Rapid measurement of scalar three-bond ¹H^N-¹H^α spin coupling constants in ¹⁵N-labelled proteins

Two new 2D NMR experiments, CT-HMQC-HA and CT-HMQC-HN, are proposed for the rapid measurement of homonuclear ³J_HNHα coupling constants of uniformly ¹⁵N-enriched proteins in solution. The experiments are based on the comparison of the signal intensities in a pair of constant-time [¹⁵N,¹H]-HMQC spectra recorded with and without decoupling of the amide proton - α proton coupling. Experimental data recorded with the 78-residue N-terminal domain of the E. coli arginine repressor (ArgR-N) and with oxidized E. coli flavodoxin (176 residues) showed good agreement with ³J^HNHα coupling constants obtained by fitting of the multiplet fine structure of the amide proton resonances or from a 3D HNHA-J experiment, respectively. Quantitative estimates for the effects from different relaxation rates of in-phase and antiphase magnetization are given.