Figure 3
From: Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation
Effects of vinculin Vd1 on talin rod refolding.
(a–b) Vinculin Vd1 does not trigger talin rod unfolding at low forces. Five representative unfolding force-extension curves were selected from >30 repeating stretch-relaxation cycles, at Vd1 concentrations of (a) 0 nM and (b) 10 nM. The curves are smoothed with a 0.05 s time window and the time trajectories of the extension change during multiple force-increase scans at a constant loading rate of 5 pN/s are shown in the top x axis. In each force-increase scan, time zero is set to be the beginning of the force scan; therefore curves from different scans can be plotted in one figure panel. (a) In the absence of Vd1, the curve shows three unfolding events during the stretch phase in each cycle. (b) After incubation with Vd1, the characteristic unfolding steps were observed in the first force-cycle stretching (red). Unfolding events were absent in subsequent force-cycles (black), indicating complete inhibition of talin rod refolding by Vd1 bound to mechanically exposed VBS in the first force-increase scan. (c–d) The effect of lower Vd1 concentrations (a) 0 nM (c) 1 nM and (d) 10 nM. For clarity the initial unfolding curve is omitted. Insets in (c) and (d) show ∼3 nm steps consistent with force-induced vinculin head dissociation at high forces (see explanation in main text). (e) Histogram of talin rod unfolding forces at different Vd1 concentrations obtained from two independent talin rod tethers, normalized by the number of stretch-relaxation force cycles. (f) R3 can be activated for Vd1 binding at less than 7 pN of force. Force cycle experiments between 1 and 7 pN for a single WT R1–R3 tether in the absence (colored traces) or in the presence (grey traces) of 10 nM Vd1.
