Conformational exchange of aromatic side chains by H 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 C CPMG relaxation dispersion experiments. Here we investigate the possibility of aromatic H CPMG relaxation dispersion experiments as a complementary method. Artifact-free dispersions are possible on uniformly H and C 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 C 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 H relaxation dispersion profiles in phenylalanine, tyrosine and the six-ring moiety of tryptophan, display anomalous behavior caused by J H-H couplings and, if present, strong C-C couplings. Therefore they require site-selective H/H and, in case of strong couplings, C/C labeling. In summary, aromatic H 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.