Fig. 5.

Stepwise stabilization between CNB domains in partial cAMP-bound states. a Representative force-extension curves of two intermediate-liganded states (A₁B₀ and A₀B₁) for the type-III regulatory subunit S110C/S376C. Zoomed-in are the unfolding trajectories of A₁B₀ compared with fully bound state (i.e., A₁B₁), showing the lack of N3A motif hopping in A₁B₀. b Unfolding force probability distribution and force-dependent folded-state lifetimes for intermediate-liganded states: CNB-A domain in A₀B₁ (top) and CNB-B domain in A₁B₀ (bottom). The corresponding isolated domains in the apo state (white bars and symbols) are shown for comparison. Solid lines are the unfolding force distribution reconstructed from force-dependent lifetimes. c WLC analysis of changes in extension upon unfolding vs. force in A₁B₀ and A₀B₁ for the CNB-A and CNB-B domains (dashed lines). Expected WLC curves are shown for CNB-A domain without the N3A motif (dark purple) and with the N3A motif (light purple). d cAMP titration plot showing the fraction of apo (A₀B₀), intermediate (A₁B₀ or A₀B₁), and fully bound (A₁B₁) species. Lines correspond to the global fit to the equations for each population species (Supplementary Methods). Error bars are the weighted SD of different single molecules. e Fractional titration plot of isolated CNB-A (top) and CNB-B (bottom) domains (type-I constructs). The error bar corresponds to the SD of five to ten different molecules