Changes in amide hydrogen solvent accessibility determined by H/H and H/D exchange with NMR.

CLEANEX-HSQC experiments for H/H measurements were collected with a mixing time of 100 ms using a recycle delay of 1.5 s. For H/D exchange, NMR samples were dehydrated at room temperature in a Savant SVC 100H Speed Vacuum Concentrator with RH 40–11 rotor and resuspended in an equivalent volume of D2O. We used a Best-TROSY HSQC experiment to assess H/D exchange. The experimental parameters for these experiments were similar to those used in the CLEANEX experiments, except that the recycle delay was reduced to 0.2 s, resulting in a total acquisition time of 13 minutes. The first Best-TROSY spectrum for each sample was taken ~30min (including the 13min collection time) after resuspension. Six sequential measurements were taken immediately to capture more rapid exchange. Additional measurements were taken over the course of 14 days for a total of 20 time points. Peak intensities were measured as heights determined through CcpNmr Analysis. CLEANEX and core residue peak intensities were normalized to respective TROSY spectra. For non-core resonances in the H/D exchange spectra, the measured intensities were normalized to an average of unaffected residue intensities. H/D exchange rates for non-core residues were calculated by fitting to an exponential decay as described previously⁴⁵.