Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach

Relaxation compensated constant-time Carr–Purcell–Meiboom–Gill relaxation dispersion experiments for amide protons are presented that detect μs-ms time-scale dynamics of protein backbone amide sites. Because of their ten-fold larger magnetogyric ratio, much shorter 180° pulses can be applied to 1H than to 15N spins; therefore, off-resonance effects are reduced and a wider range of effective rf fields can often be used in the case of 1H experiments. Applications to [1H-15N]-ubiquitin and [1H-15N]-perdeuterated HIV-1 protease are discussed. In the case of ubiquitin, we present a pulse sequence that reduces artifacts that arise from homonuclear 3J(HN-Hα) coupling. In the case of the protease, we show that relaxation dispersion of both 1H and 15N spins provides a more comprehensive picture of slow backbone dynamics than does the relaxation dispersion of either spin alone. We also compare the relative merits of 1H versus 15N transverse relaxation measurements and note the benefits of using a perdeuterated protein to measure the relaxation dispersion of both spin types.