Fig. 4. Untying and tying the knotting loop of TrmD Δhelix45,128. (A) Schematics of stretching protein TrmD Δhelix45,128. The stretching sites are colored in grey. (B) Representative stretching–relaxation curves of TrmD Δhelix45,128 showing distinct unfolding behavior of the same molecule. Relaxation of the unfolded protein led to a “refolding” event at ∼4 pN. However, it remained unknown whether the protein folded into its native state with the trefoil knot. In the subsequent stretching force–distance curves, either a typical two-state unfolding at a force of ∼7 pN or a “hump” feature was observed. (C) Histograms of unfolding and “refolding” forces of TrmD Δhelix45,128 at a pulling speed of 50 nm s−1. Inset: force-dependency of the unfolding/folding rate constants. Dotted lines are fits of the experimental data to the Bell–Evans model. (D) Relationship of the knotting probability versus Δt at zero force. The dotted line is the fit of the experimental data to the first order rate law, yielding a knotting rate constant of (3.8 ± 1.0) × 10−2 s−1.
