Fig. 4.

The CNB-B domain bound to cAMP is required for the N3A motif to fold. a (1) The regulatory subunit unfolds (red) and (2) refolds (blue), revealing the first reversible transition corresponding to the N3A motif (orange arrow). (3) In the following cycle, the regulatory subunit was stretched until the N3A motif and the CNB-B domain unfold, whereas the CNB-A domain remains folded. (4) The force was decreased to 4 pN, a force that does not allow the CNB-B domain to refold. (5) The force was then oscillated between 6 and 15 pN for several cycles (~20) to test whether the N3A motif was able to refold, while the CNB-B domains remain unfolded. (6) The force was increased to 20 pN to unfold the CNB-A domain. (7) The force was decreased to 1 pN, allowing the complete protein to refold and begin another set of experiments. The trajectories in gray represent the unfolding pathways from the immediately previous cycle, thereby serving as reference on the progression of experiment. b Pairwise contact map comparing the interaction established by the N3A motif in the regulatory subunit of PKA (left). The contacts established by the N3A motif were obtained using a 8 Å cutoff (right): (1) contacts established by residues within the N3A motif; (2) contacts between the N3A motif and the B/C helix; and (3) contacts between the N3A motif and the CNB-B domain. Cartoons rendering the three sets of contacts are shown next to the contact map. Residues in the N3A motif are colored in yellow, in the B/C helix in red, and in the CNB-B domain in blue