APPLICATION OF MULTIPLE-QUANTUM LINE NARROWING WITH SIMULTANEOUS H-1 AND C-13 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 s calar couplings to establish connectivities among backbone and/or side -chain nuclei. In medium-sized (15-30 kDa) proteins, short transverse relaxation times of C-alpha single-quantum states limit signal-to-nois e (S/N) ratios. These relaxation properties can be improved using hete ronuclear multiple-quantum coherences (HMQCs) instead of heteronuclear single-quantum coherences (HSQCs) in the pulse sequence design. In sl owly tumbling macromolecules, these HMQCs can exhibit significantly be tter transverse relaxation properties than HSQCs. However, HMQC-type e xperiments 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 exp eriments using simultaneous H-1 and C-13 constant-time (CT) periods to eliminate the t(1) dependence of these scalar coupling effects. hese simultaneous CT PFG-(HA)CANH and PFG-(HA)CA(CO)NH HMQC-type experiment s exhibit sharper resonance line widths and often have better S/N rati os than the corresponding HSQC-type experiments. Results on proteins r anging in size from 6 to 30 kDa show average methine (CH)-H-alpha HMQC :HSQC enhancement factors of 1.10 +/- 0.15, with about 40% of the cros s peaks exhibiting better S/N ratios in the simultaneous CT-HMQC versi ons compared with the HSQC versions.