(H)NCAHA and (H)CANNH experiments for the determination of vicinal coupling constants related to the ϕ-torsion angle

Abstract

A set of three-dimensional triple-resonance experiments is described which provide \({}^{\text{3}}{\text{J}}_{{\text{H}}^{\text{N}} {\text{H}}^\alpha } \) , \({}^{\text{3}}{\text{J}}_{{\text{H}}^{\text{N}} {\text{CO}}} \) , \({}^3J_{H^N C^\beta } \) and \({}^{\text{3}}{\text{J}}_{{\text{H}}^\alpha {\text{CO}}} \) coupling constants. The pulse sequences generate E.COSY-like multiplet patterns and comprise a magnetization transfer from the amide proton to the α-proton or vice versa via the directly bound heteronuclei. For residues with the ¹H^α spin resonating close to the H₂O signal, a modified HNCA experiment can be employed to measure the vicinal ¹H^N,¹H^α couplings. Ambiguities associated with the conversion of \({}^{\text{3}}{\text{J}}_{{\text{H}}^{\text{N}} {\text{H}}^\alpha } \) values into ϕ-angle constraints for protein structure determination can be resolved with the knowledge of the heteronuclear ³J-couplings. In favourable cases, stereospecific assignments of glycine α-protons can be obtained by employing the experiments described here in combination with NOE data. The methods are applied to flavodoxin from Desulfovibrio vulgaris.