Supplementary Figure 6: Force-clamp experiment for detection of single disulfide reduction events

(a) Schematics of the experiment. Disulfide bonded domains (red) show a 2 two-step unfolding pattern. The first step (~12 nm) corresponds to the unfolding of the beta sheets that are not trapped in the disulfide bond while the second step (~15 nm) shows the unfolding of the rest of the protein after the reduction of the disulfide bond cause by thioredoxin. Not disulfide bonded domains have a single-step (~27 nm) unfolding pattern that represents the stretching of the whole domain. (b) Experimental trace in the absence of thioredoxin enzymes of LSCA titin with seven steps representing each immunoglobulin domain visualized within the first pulse at 135 pN (grey). The fully unfolded domains are marked with an arrow (~27 nm) whereas disulfide bonded domains are marked with an asterisk (~5-20 nm). No steps are detected in the second stage at 80 pN (green) that was kept for 20 s. (c) Two populations of steps can be observed in the histogram (n=286). The histogram below shows step captured in the 80 pN pulse where reduction are generally observed in the presence of Trx (Fig. 3 in main text). (d) and (e) Experimental trace and steps histograms (n=132) for human titin, respectively. In this case eight unfolding events are shown from which only one shows step size corresponding to a disulfide bonded domain. In the traces from human titin is common to observe 1 or 2 disulfide bonded domains. In both cases the histograms of the unfolding pulse resemble the histograms of the force-extension experiments, as expected.