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 mus-ms time-scale dynamics of protein backbone amide sites. Because of their ten-fold larger magnetogyric ratio, much shorter 180degrees pulses can be applied to H-1 than to N-15 spins; therefore, off-resonance effects are reduced and a wider range of effective rf fields can often be used in the case of H-1 experiments. Applications to [H-1-N-15]-ubiquitin and [H-1-N-15]-perdeuterated HIV-1 protease are discussed. In the case of ubiquitin, we present a pulse sequence that reduces artifacts that arise from homonuclear (3)J(H-N-H-alpha) coupling. In the case of the protease, we show that relaxation dispersion of both H-1 and N-15 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 H-1 versus N-15 transverse relaxation measurements and note the benefits of using a perdeuterated protein to measure the relaxation dispersion of both spin types.