FIGURE 3.

Mechanical unfolding exposes disulfide 57–146 enabling reduction by TCEP. Mechanical unfolding of hSOD1 and cleavage of its disulfide bond were monitored using force-clamp spectroscopy. Single I27₃-SOD-I27₃ polyproteins were initially subject to a linear increase in force from 0 to 250 pN at 50 pN/s, and then force was held constant at 250 pN. The recordings presented in all panels were obtained in a solution containing 2.5 mm TCEP. Mechanical force triggers mechanical unfolding of the protein modules in the polyprotein. I27 acts as a fingerprint protein that allows us to select traces where hSOD1 has been subjected to force. Mechanical unfolding of hSOD1 renders the disulfide accessible to TCEP, and enables thiol/disulfide exchange reactions involving the disulfide in hSOD1. In panels A, B, and C, the lower trace shows the force pulse, while the upper traces correspond to the length of the polyprotein. Each I27 unfolding event produces a 25-nm step (black dots). Occasionally, we detect unfolding of hSOD1 at low forces (marked by gray dots). An example of each one of the three patterns of steps that we found is shown in A through C. A, after extending the six I27 modules, an additional extension of 31 nm (red) is observed; B, after the extension of six I27 modules, two additional steps of 19 nm (blue) and 12 nm (green) are detected; C, the 12-nm step appears before the 19-nm step. D, distribution of step sizes induced by the mechanical extension of I27₃-SOD-I27₃ in the presence of TCEP. To build the histogram, we pooled results obtained at different concentrations of TCEP. We did not include in the histogram steps of 25 nm coming from the extension of I27 modules. The distribution reveals four peaks centered at: 12 nm (green), 19 nm (blue), 22 nm (gray), and 31 nm (red). The rest of the steps recorded. which could not be assigned and probably represent nonspecific interactions, are shown in white. n = 871 events.