Figure 3. Oxidoreductase Mediated Disulfide Cleavage and Reformation under Force.

(A) After full unfolding of eight I27ox domains (11.4-nm steps), PDI^RED was added to the flow cell, causing eight distinct disulfide cleavage events (14.6-nm steps). After quenching the force to 5 pN, a downward folding staircase is observed. Subsequent unfolding at 84 pN reveals a mixture of 11- and 14-nm steps, indicating reformation of the disulfide bond.

(B) Disulfide reformation could also be achieved with oxidized PDI. After full unfolding of eight I27^OXD domains, TCEP (10 mM) was added to the flow cell, resulting in eight 14.6-nm steps due to the reduction of the disulfide bond. Quenching the force to 5 pN resulted in only a single refolding event. The protein was stretched again at high force to remove the TCEP with 53 washes using buffer. Reducing the force to 2 pN caused the refolding of 73 × I27^RED domains, as indicated by the 25-nm steps. After full unfolding, PDI^OXD was added to the flow cell, and the force was reduced to 5 pN again. In the presence of the oxidoreductase, a folding staircase is again observed. Subsequent pulling at 84 pN demonstrates 73 × 11- and 14-nm steps, indicating nearly complete reformation of the disulfide bond at 5 pN. Histograms in (A) and (B) are pooled from all of the oxidative folding experiments performed with the PDI oxidoreductase.