Figure 6.

Interrupted binding experiments. A, schematic of the double-jump setup. In the first jump, 4 μm PSG was mixed with 8 μm D-CRIPT₆ (i.e. the final concentrations after mixing were 2 μm and 4 μm, respectively) and incubated for a certain delay time in an aging loop. Following the set delay time, any formed complex was dissociated by a large excess of unlabeled CRIPT in a second jump (final concentrations were 1 μm PSG, 2 μm D-CRIPT₆, and 50 μm unlabeled CRIPT). B, the dissociation kinetics were monitored in the flow cell. The kinetic traces were fitted simultaneously to a double-exponential function with either locked k_obs values (the two average k_off^app values from single-jump experiments, 1.35 s⁻¹ and 0.17 s⁻¹, respectively) or shared and free-fitted k_obs values (k_off1^app = 2.1 s⁻¹ and k_off2^app = 0.35 s⁻¹). The best fit curves in the figure correspond to the latter fit. C and D, the kinetic amplitudes from the respective fits in B were plotted against delay time. This reflects the build up of PSG_A:D-CRIPT₆ and PSG_B:D-CRIPT₆, respectively, with time. These amplitude data, in turn, were fitted to a double-exponential function to obtain two “observed double-jump rate constants” for the build up of PSG_A and PSG_B, respectively. From the fit, k_obs^DJ values of around 20–40 s⁻¹ and 0.1–0.8 s⁻¹, respectively, were obtained. Although the parameters are underdetermined, it is clear that there is an initial increase in the respective population, followed by a decrease in PSG_A:D-CRIPT₆. The expected concomitant increase in PSG_B:D-CRIPT₆ is lost in the experimental noise.