Conformational exchange of aromatic side chains by ¹H CPMG relaxation dispersion

Aromatic side chains are attractive probes of protein dynamics on the millisecond time scale, because they are often key residues in enzyme active sites and protein binding sites. Further they allow to study specific processes, like histidine tautomerization and ring flips. Till now such processes have been studied by aromatic ¹³C CPMG relaxation dispersion experiments. Here we investigate the possibility of aromatic ¹H CPMG relaxation dispersion experiments as a complementary method. Artifact-free dispersions are possible on uniformly ¹H and ¹³C labeled samples for histidine δ2 and ε1, as well as for tryptophan δ1. The method has been validated by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined rate constants and populations agree well with previous results from ¹³C CPMG relaxation dispersion experiments. The CPMG-derived chemical shift differences between the folded and unfolded states are in good agreement with those obtained directly from the spectra. In contrast, the ¹H relaxation dispersion profiles in phenylalanine, tyrosine and the six-ring moiety of tryptophan, display anomalous behavior caused by ³J ¹H-¹H couplings and, if present, strong ¹³C-¹³C couplings. Therefore they require site-selective ¹H/²H and, in case of strong couplings, ¹³C/¹²C labeling. In summary, aromatic ¹H CPMG relaxation dispersion experiments work on certain positions (His δ2, His ε1 and Trp δ1) in uniformly labeled samples, while other positions require site-selective isotope labeling.