Fig. 3
Mechanical characterization of I69oxidized identifies disulfide isomerization as a contributor to titin elasticity. Diagrams on the left describe the mechanism of extension of I69 due to unfolding a, b and unfolding-triggered redox reactions c, d. Each pathway is illustrated with a trace and a histogram of step sizes. Theoretical step sizes are indicated by dashed lines (Supplementary Note 2). a The unfolding of (I69reduced)8 pulled at 170 pN results in a uniform step-wise extension of the polyprotein (26 nm steps in the blue trace). Similar to the trace shown in Fig. 2f, the absence of steps in the probe pulse indicates low refolding kinetics of I69reduced (Δt = 5 s). b When (I69oxidized)8 is stretched at 100 pN, a predominant population of 6 nm emerges in the first 3 s, marking the unfolding of I69 domains containing disulfide CysB–CysG. c After 3 s (gray part of the trace in (b)), two new populations of 4 and 17 nm steps appear. Mechanical unfolding activates a reaction of disulfide exchange where the thiol group of CysF can attack either the sulfur of CysB or CysG, giving rise to the 4 and 17 nm step populations. d In the presence of 10 mM Tcep, additional 19 nm steps are detected (Gaussian fit centered at 19.2 ± 0.3 nm), marking the reduction of disulfide CysB–CysG by Tcep (black sphere). The reduction of the newly interchanged disulfide CysB–CysF and CysF–CysG along the trajectory produces steps of 3 and 16 nm22
