Fig. 3.
Quantification of the F,S exchange process. (a) Longitudinal exchange spectroscopy recorded on WT ClpP at 0.5°C. The decay of autopeaks and buildup of cross-peaks is illustrated, where I/I0 is the intensity of a given correlation normalized to the maximum intensity of the appropriate autopeak at the shortest time point used, uncorrected for any relaxation during the pulse scheme (see Materials and Methods). Experimental data points are indicated with “x,” and the dotted and solid lines correspond to the best fits of the data for states F and S, respectively (all curves are fitted simultaneously). Because of spectral overlap, the autopeak derived from the I151 F state could not be quantified. (b) Methyl TROSY relaxation dispersion profiles recorded at 0.5°C. Contributions to relaxation rates from carbon chemical shift anisotropy as well as differences in 1H chemical shifts between states result in differences in plateau values for the curves associated with a given state (F or S) at 600 and 800 MHz. (c) Determination of the activation energies that characterize the F,S exchange process. Note that kF→S ≈ kS→F so that the activation parameters are very similar for F→S and S→F. The relaxation dispersion profile of I149 recorded at 40°C is shown in Inset.