Fig. 2
Disulfide bonds in I65–I70. a Schematic representation of cysteine position in the six Ig domains composing I65–I70, and X-ray structure of I67 (pdb: 3B43) with arrows representing the applied force at the termini in AFM experiments. CysB, CysF and CysG are highlighted. Step sizes are indicated in the diagrams. We fitted Gaussian curves to all histograms presented here to estimate the different populations of observed step sizes. b Typical trace recorded applying a constant force of 170 pN on I65–I70reduced. c The histogram of step sizes shows that only reduced domains are detected (unfolding step of 26 nm). d Stretching I65–I70oxidized at 100 pN produces new distinct populations of shorter steps, with a different kinetics of appearance. The first 3 s of the experiment are shown in red. In this experimental recording, the polyprotein extends over time in steps of 26, 10, 6, 6 and 10 nm and in a last 4 nm step (in light gray, a 15 s portion of the trace has been cut out). e Histograms of the step sizes obtained with I65–I70oxidized (red in the first 3 s and gray for delayed events). The I65–I70oxidized unfolds showing two large populations of 6 and 10 nm steps in agreement with the unfolding and extension of domains shortened by disulfides CysB–CysG and CysB–CysF, respectively. The presence of longer 26 nm steps illustrates that some domains remain reduced. The steps at 4 and 17 nm correspond to events of disulfide isomerization. f This experimental trace obtained with a typical three-pulse protocol allows the evaluation of the refolding ability of I65–I70reduced. All domains mechanically unfold during a first pulse (unfolding pulse), then collapse when the force is switched off for a set time Δt (quench). The third pulse (probe) probes the domains that recovered their native state during the quench phase. In this trace, the absence of steps in the probe pulse indicates that none of the mechanically unfolded domains refolded during the quench (Δt = 10 s)
