Cross-Correlated Relaxation Enhanced 1H−13C NMR Spectroscopy of Methyl Groups in Very High Molecular Weight Proteins and Protein Complexes

Abstract

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 ClpP, 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.