Fig. 4. An RNC-NAC binding assay shows co-binding of SRP and NAC on the RNC.

a Inset: scheme of the FRET assay to measure RNC-NAC binding. RNC was labeled with BDP (cyan star) at the N-terminus of signal sequence, and NAC was labeled with Cy3B (green star) at residue 57 of NACβ. Figure: Fluorescence emission spectra showing FRET between labeled RNC and NAC. Where indicated, the reactions contained 1 nM RNC(ss)^BDP or 15 nM unlabeled RNC(ss) and 5 nM NAC^Cy3B. The spectra were measured by excitation at 485 nm. b Competition assay to determine the RNC binding affinity of unlabeled NAC and NACmt. 1 nM RNC^BDP and 20 nM NAC^Cy3B were pre-incubated to form the RNC^BDP-NAC^Cy3B complex, followed by addition of unlabeled NAC or NACmt to compete with NAC^Cy3B for RNC binding. The data with NAC were fit to Eq. (6) in the Methods, which gave K_d values of 0.55 and 0.50 nM for the binding of unlabeled NAC to RNC(ss) and RNC(ssmt), respectively. c, d Equilibrium titrations to measure the binding of NAC to RNC(ss) (c) and RNC(ssmt) (d) in the presence of increasing SRP concentrations. The reactions contained 5 nM RNC^BDP and the indicated concentrations of NAC^Cy3B, unlabeled NAC and SRP. The data were fit to Eq. (5) to obtain apparent K_d,NAC values at the individual SRP concentrations. e, f Comparison of the experimentally determined apparent K_d,NAC values at different SRP concentrations (black squares) with predictions from the anti-cooperative model in Fig. 3g (blue line, simulated using Eq. (10)) and the competitive model in Supplementary Fig. 4a (red line, simulated using Eq. (11)). The simulations in e used K_d,SRP, K_d,NAC and α values of 0.36 nM, 1.4 nM, and 6.6, respectively. The simulations in f used K_d,SRP, K_d,NAC and α values of 1.5 nM, 1.2 nM, and 1.4, respectively. The experimental data were shown as mean ± SD, with n = 3 independent measurements on the same biological sample. Source data for a–f are provided in the Source Data file.