1H R1ρ relaxation dispersion experiments in aromatic side chains

Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic ¹H R_1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated ¹H–¹³C spin pairs with vicinal ²H–¹²C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the ³J_HH couplings and limited chemical shift differences among ¹H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, k_flip, agree well with previous results from ¹³C R_1ρ relaxation dispersion experiments, and yield ¹H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic¹H R_1ρ relaxation dispersion experiment in combination with the site-selective ¹H–¹³C/²H–¹²C labeling scheme enable measurement of exchange rates up to k_ex = 2k_flip = 80,000 s^–1, and serve as a useful complement to previously developed ¹³C-based methods.