Site-selective ¹H/²H labeling enables artifact-free ¹H CPMG relaxation dispersion experiments in aromatic side chains

Aromatic side chains are often key residues in enzyme active sites and protein binding sites, making them attractive probes of protein dynamics on the millisecond timescale. Such dynamic processes can be studied by aromatic ¹³C or ¹H CPMG relaxation dispersion experiments. Aromatic ¹H CPMG relaxation dispersion experiments in phenylalanine, tyrosine and the six-ring moiety of tryptophan, however, are affected by ³J ¹H-¹H couplings which are causing anomalous relaxation dispersion profiles. Here we show that this problem can be addressed by site-selective ¹H/²H labeling of the aromatic side chains and that artifact-free relaxation dispersion profiles can be acquired. The method has been further validated by measuring folding-unfolding kinetics of the small protein GB1. The determined rate constants and populations agree well with previous results from ¹³C CPMG relaxation dispersion experiments. Furthermore, the CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained directly from the spectra. In summary, site-selective ¹H/²H labeling enables artifact-free aromatic ¹H CPMG relaxation dispersion experiments in phenylalanine and the six-ring moiety of tryptophan, thereby extending the available methods for studying millisecond dynamics in aromatic protein side chains.