Fig. 2: Association/dissociation kinetics are highly protein concentration-dependent.

a–c Overlay of transfer efficiency histograms of 50 pM ProTα E56C/D110C labeled with Alexa Fluor 488/594 at different concentrations of unlabeled H1 (10 nM, 1 μM, and 20 μM) titrated with increasing concentration of unlabeled ProTα exhibit a transition from slow to fast exchange between the populations (see legends for color code). The mean transfer efficiency of bound ProTα decreases slightly with increasing H1 concentration owing to the formation of higher-order complexes (Supplementary Fig. 3d). d ¹H–¹⁵N-HSQC spectra of 20 μM ¹⁵N-ProTα (recorded at 750 MHz and 200 mM ionic strength) titrated with increasing concentrations of unlabeled H1 (see legend for color code). e Examples of individual resonances illustrate the continuous shifting of a single population-averaged peak. f 1D ¹⁵N projections of the NMR resonances at different concentrations of unlabeled H1. g, h Comparison of NMR 1D ¹⁵N lineshapes calculated using the Bloch–McConnell equation for a two-state (g) or a four-state binding mechanism (h) (see Supplementary Table 2 for rate coefficients and “Methods” for details).