Multi-probe relaxation dispersion measurements increase sensitivity to protein dynamics

Abstract

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion measurements are a valuable tool for the characterization of structural transitions on the micro–millisecond timescale. While the measurement of ¹⁵N relaxation dispersion is now routine, the measurements with alternative nuclei remain limited. Here we report ¹⁵N as well as ¹H R₂ relaxation dispersion measurements of the N23PP/S148A “dynamic knockout” mutant of dihydrofolate reductase. The ¹H dispersion measurements are complementary to ¹⁵N data as many additional residues are observed to have dispersive behavior for the ¹H nucleus. Simultaneous fitting of the dispersion profiles for the two nuclei increases the accuracy of exchange parameters determined for individual residues and clustered groups of residues. The different sensitivity of the two nuclei to changes in backbone torsional angles, ring currents, and hydrogen bonding effects provides important insights into the nature of the structural changes that take place during the exchange process. We observe clear evidence of direct and indirect hydrogen bond effects for the ¹⁵N and ¹H chemical shift changes in the active-site, modulation of ring current shielding in the CD-loop and backbone torsional changes in a cluster of residues associated with the C-terminus. This work demonstrates the power of combined ¹H and ¹⁵N probes for the study of backbone dynamics on the micro-millisecond timescale though the analysis of chemical shift changes.