Application of multiple-quantum line narrowing with simultaneous 1H and 13C constant-time scalar-coupling evolution in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments

Many triple-resonance experiments make use of one-bond heteronuclear scalar couplings to establish connectivities among backbone and/or side-chain nuclei. In medium-sized (15-30 kDa) proteins, short transverse relaxation times of Cα single-quantum states limit signal-to-noise (S/N) ratios. These relaxation properties can be improved using heteronuclear multiple-quantum coherences (HMQCs) instead of heteronuclear single-quantumcoherences (HSQCs) in the pulse sequence design. In slowly tumbling macromolecules, these HMQCs can exhibit significantly better transverse relaxation properties than HSQCs.However, HMQC-type experiments also exhibit resonance splittings due to multiple two- and three-bond homo- and heteronuclear scalar couplings. We describe here a family of pulsed-field gradient (PFG) HMQC-type triple-resonance experiments using simultaneous 1H and 13C constant-time (CT) periods to eliminate the t1 dependence of these scalar coupling effects. These simultaneous CT PFG-(HA)CANH and PFG-(HA)CA(CO)NH HMQC-type experiments exhibit sharper resonance line widths and often have better S/N ratios than the corresponding HSQC-type experiments. Results on proteins ranging in size from 6 to 30 kDashow average methine CαH HMQC:HSQC enhancement factors of 1.10 ± 0.15, with about 40% of the cross peaks exhibiting better S/N ratios in the simultaneous CT-HMQC versions compared with the HSQC versions