Fig. 2. Stretching TrmD9,246 to a tightened trefoil knot involves two parallel pathways. (A) Representative force–distance curves showing two-state unfolding of TrmD9,246. The pulling speed was 50 nm s−1. Inset: representative force–distance curves showing the unfolding of full-length TrmD9,246–(NuG2)2. The mechanical unfolding events of the fingerprint domains NuG2 are colored in black and indicated with circles, and the unfolding events of TrmD9,246 are indicated with squares. (B) Representative force–distance curves of TrmD9,246via the three-state unfolding pathway. The pulling speed was 50 nm s−1. The zoom-in view showed that the protein unfolds via an intermediate state. The protein unfolding from N → I and I → U is colored in blue and green, respectively. The dashed lines in (A) and (B) are pseudo WLC fits to the force–distance curves. The fits were primarily used for identification of various states (folded, intermediate and unfolded state) of protein in the force–distance curves, and the fitting parameters were not physically meaningful. (C) Force–extension relationships of TrmD9,246. Extension is the length increase at a given force upon unfolding. Error bars correspond to the standard deviation of the data measured from different molecules. Dashed lines are WLC fits to the force–extension data measured for N → I (blue), I → U (green) and N → U (red), respectively. The WLC fits yielded a persistence length of 0.8 nm and a ΔLc1 of 9.8 ± 0.4 nm, ΔLc2 of 28.9 ± 1.0 nm and ΔLc of 36.8 ± 1.8 nm, respectively. (D) Unfolding force histograms of TrmD9,246 at a pulling speed of 50 nm s−1. Inset: force-dependency of unfolding rate constants measured by the model-free method proposed by Oesterhelt et al. Fitting to the Bell–Evans model yielded the intrinsic unfolding rate constant α0 and the unfolding distance Δxu, as summarized in Table S1.†.
