Supplementary Figure 7: Supporting information for the kinetic simulations, and additional data for chase experiments of the RNCRodZ–SecA complex.
From: The molecular mechanism of cotranslational membrane protein recognition and targeting by SecA
a, Measurement of the association rate constant of the SecA–RNCRodZ complex. 10 nM Cm-labeled RNCRodZ was mixed with indicated concentrations of BDP-labeled SecA, and the fluorescence change was fit to Equation 3 in Supplementary Note 1 to extract the observed association rate constant (kobsd). The value of kobsd was plotted against SecA concentration and fit to Equation 7 in Supplementary Note 1 to determine k-1. b, Equilibrium measurement of the transfer reaction. 10 nM at indicated chain lengths was pre-incubated with 30 nM BDP-labeled SecA. Increasing amounts of SecYEG-Nd were then added to the preformed complex, and the increase in Cm fluorescence due to the loss of FRET was monitored. The data were fit to Equation 8 in Supplementary Note 1 and gave a K1/2 value of 45 ± 18 nM, 29 ± 5.4 nM, and 13 ± 2.1 nM for RNCRodZ at nascent chain lengths of 91, 131, and 171aa, respectively. c–f, Changes in the rate constants of RNCRodZ–SecYEG association (k2) and dissociation (k-2) (the Kd value for RNCRodZ–SecYEG was held constant) do not affect the kinetics behavior for both the passive (c,d) and active (e,f) models. g, Reaction scheme (left) and simulation (right) of the experiments to measure the dissociation rate constant (k1) of SecA from RNCRodZ. A preformed complex of Cm (blue star)-labeled RNCRodZ with BDP-labeled SecA was chased with excess unlabeled SecA to initiate complex dissociation, and the loss of FRET was monitored in real time. h,i, Representative time courses for measurement of k1 at nascent chain lengths of 91aa (h) and 131aa (i). The data were fit to Equation 3. All traces are the average of 6–8 measurements. j,k, Representative fluorescence time traces for chase of SecA-bound RNCRodZ complex with SecYEG-Nd (j) or empty nanodisc (k) at a nascent chain length of 131aa. Reactions were carried out and analyzed as in Supplementary Fig. 6c,d, and the obtained rate constants are summarized in Fig. 6f. Note that the time traces are biphasic, and control experiment indicated that the slow phase was due to dye bleaching (see Methods).
