1H R1Ï? relaxation dispersion experiments in aromatic side chains
 

1H R1Ï? relaxation dispersion experiments in aromatic side chains

Abstract

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 R1Ï? 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 1Hâ??13C spin pairs with vicinal 2Hâ??12C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmannâ??Hahn matching due to the 3JHH 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, kflip, agree well with previous results from 13C R1Ï? 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 R1Ï? relaxation dispersion experiment in combination with the site-selective 1Hâ??13C/2Hâ??12C labeling scheme enable measurement of exchange rates up to kexâ??=â??2kflipâ??=â??80,000Â*sâ??1, and serve as a useful complement to previously developed 13C-based methods.



Source: Journal of Biomolecular NMR