Sensitivity Enhancement of Triple-Resonance Protein NMR Spectra by Proton Evolution of Multiple-Quantum Coherences Using a Simultaneous ¹H and ¹³C Constant-Time Evolution Period

Short transverse relaxation times of Cα and Hα single-quantum states in proteins reduce signal-to-noise ratios of heteronuclear correlation experiments involving transfers of Cα and Hα coherences. To overcome this “short transverse relaxation problem”, we have developed a simultaneous 1H and 13C constant-time (sim-CT) heteronuclear multiple-quantum coherence (HMQC) scheme. New features in this design include: (i) utilization of heteronuclear multiple-quantum coherences for better transverse relaxation properties, (ii) concatenation of proton evolution into the simultaneous 1H and 13C constant-time period to eliminate separate time periods for proton evolution, and (iii) use of simultaneous 1H and 13C constant-time to remove resonance splitting due to multiple two- and three-bond homo- and heteronuclear scalar couplings. This general approach for sensitivity enhancement is demonstrated for the HA(CA)(CO)NH triple-resonance experiment. Results on proteins show that, compared with the heteronuclear single-quantum coherence version of the same experiment, on average the sim-CT HMQC version of HA(CA)(CO)NH exhibits enhancements of ∼20%.