Fig. 2. Mechanical unfolding of the mutated C1 domain without the ester bond.

a Thr-11 of C1 was mutated to Ala to eliminate the ester bond to yield the C1_T11A mutant. b Topology of C1_T11A. The ester bond cannot form in the mutant. c Representative single-molecule force–extension curve of stretching Fgβ-(GB1)₂-C1_T11A-(GB1)₂-cys following the same experimental protocol shown in Fig. 1d. Each peak was fitted by worm-like chain (WLC) model of polymer elasticity. The peak with a ΔL_c of 47 nm corresponds to the unfolding of C1_T11A, the next four peaks correspond to the unfolding of GB1 domains, and the last peak corresponds to the rupture of the Fgβ/SdrG complex. d Histogram of contour length increment for C1 unfolding is centered ~47 nm. e Unfolding force histogram of C1 domain at a pulling speed of 1.6 μm s⁻¹ measures an average unfolding force of 92 ± 41 pN (n = 129, total number of C1 unfolding events). Fitting the force distribution with Monte Carlo simulation⁶⁵ results in a Δx_u of 0.27 nm and α₀ of 0.35 s⁻¹ (Supplementary Fig. 8). This experiment was repeated three times independently with similar results. Source data are provided as a Source Data file.