Fig. 5. Mechanical unfolding of C1-df.
a Schematic of the AFM-based single-molecule force spectroscopy experiments. Polyprotein Fgβ-(GB1)2-C1-df-(GB1)2-Spytag was linked to the cys-GB1-Spycatcher modified substrate covalently through the Spycatcher/Spytag chemistry and picked up by the SdrG-cys modified cantilever through the Fgβ/SdrG interaction. Thus, the force–extension curves should contain five GB1 fingerprints. b Three possible unfolding/hydrolysis pathways of C1-df under load and the corresponding contour length change. c Representative force–extension curves of the “Disulfide unformed” group. Two populations of unfolding events as showed, five peaks in black related to GB1 unfolding and the purple one with ΔLc of 47 nm corresponds to the unfolding of C1-df. d Representative force–extension curves of the “Disulfide unruptured” group. The traces show only five GB1 unfolding events without the signature of C1-df unfolding or disulfide bond rupture. e Representative force–extension curves of the “Disulfide ruptured” group. Except for the five GB1 unfolding events, there is an additional peak with ΔLc of 41 nm (colored in orange) corresponding to the rupture of the disulfide bond. f 2D bar shows the relative populations of the three kinds of events without (left, n = 193) or with (right, n = 389) 10 mM TCEP. This experiment was repeated 4 times independently with similar results. Source data are provided as a Source Data file.