Cross-correlated relaxation enhanced 1H-13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes

A comparison of HSQC and HMQC pulse schemes for recording 1H-13C correlation maps of protonated methyl groups in highly deuterated proteins is presented. It is shown that HMQC correlation maps can be as much as a factor of 3 more sensitive than their HSQC counterparts and that the sensitivity gains result from a TROSY effect that involves cancellation of intra-methyl dipolar relaxation interactions. 1H-13C correlation spectra are recorded on U-[ 15N,2H], Ileδ1-[13C,1H] samples of (i) malate synthase G, a 723 residue protein, at 37 and 5°C, and of (ii) the protease CIpP, comprising 14 identical subunits, each with 193 residues (305 kDa), at 5°C. The high quality of HMQC spectra obtained in short measuring times strongly suggests that methyl groups will be useful probes of structure and dynamics in supramolecular complexes.