Fig. 4.

Folding is drastically slowed at low forces through the occurrence of misfolded intermediates. (A) Double-jump assay: (Left) Force vs. time trace while alternating between two trap distances. (Upper Right) Contour length vs. time trace of the high-force phases merged together. The vertical blue dotted lines mark the positions where a low-force phase was cut out. The trace is colored according to an HMM state assignment (details in SI Appendix). The gray colored data could not be classified unambiguously. (Lower Right) Zoom into the low-force region. For comparison, the colored dashed lines mark the contour length levels of the folding intermediates from Fig. 3. (B) Merged high-force phases of contour length vs. time traces obtained in double-jump experiments. The refolding force bias during the corresponding low-force phases was set to 0.3 pN (upper trace) and 8 pN (lower trace), respectively. DEX concentration was 200 µM. (C) Force dependence of the overall folding + binding rates $k_{FB}$ from double-jump (blue circles) experiments and passive-mode (blue squares) measurements (as presented in Fig. 3B). The error bars reflect SEM. Black dotted line shows the expected force-dependent folding + binding rates $k_{FB}$ calculated from the force-dependent kinetics as obtained from the equilibrium high-force folding measurements shown in Fig. 3 (example data in SI Appendix, Fig. S4B). The green dashed line shows a similar calculation including the possibility of misfolding branching from the IM1 state as depicted in E (details in SI Appendix). (D) Average contour length evolution for refolding against a force bias of 6.1 pN. The dashed colored lines show simulations of different models each assuming misfolding branching off a different on-pathway intermediate (colored accordingly; see text for details). Dotted colored lines mark contour length levels of the respective folding intermediates. (E) Model for folding and binding including an off-pathway misfolded structure.