Fig. 4.

Insertion of lobe II of EcNBD can create a folding-competent mtNBD structure by acting as a folding nucleus. (A) Force-extension traces of mtNBD at a pulling velocity of 500 nm/s. Shown are only stretching cycles. First, natively folded mtNBD unfolds, followed by the unfolding of weakly formed structures that do not fold back to the native fold even after 10 min. (B) Scatter plot of force vs. contour length shows the broad scattering of refolding intermediates for mtNBD (green). To compare, the events for EcNBD are shown in red and orange (RFI1 and RFI2). (C) Design of the 50% mtNBD chimeric protein consisting of lobe II from EcNBD, lobe I, and C-terminal helix from mtNBD. (D) Force-extension stretching traces at a pulling speed of 200 nm/s show the characteristic refolding events of lobe II. After ∼230 s, the chimeric protein can refold completely to its stable native fold.