Fig. 5
From: Mechanical architecture and folding of E. coli type 1 pilus domains
Oxidative folding of FimG in the presence of DsbA and FimC. a FimG construct, periplasmic FimC, and DsbA oxidoreductase. b Force-clamp trace of FimG in the presence of DsbA. A 4-pulse protocol was applied to the protein. First, 160 pN for 2 s (I91 fingerprint detection); second, force was increased to 300 pN for 5 s (or 7 s) for FimG unfolding. Third, force was quenched for 2 s to 100 pN in order to ease the reduction of the protein by DsbA. Fourth, force was quenched to 0 pN for 45 s for refolding. In order to test if FimG refolded and if its disulfide bond was reformed, we applied the same pulse protocol. I91 unfolding steps appear as a 25 nm increase in length, while oxidized FimG yields a 33 nm step size increase, and its reduction produces a 12 nm increment in length. c Refolding probability of FimG after 45 s of quenching time under different conditions (FimGSH-SH, n = 16; FimGSH-SH + FimC, n = 14; FimGSH-SH + DsbA, n = 16; FimGSH-SH + FimC + DsbA, n = 21). d Force-clamp trace for FimG in the presence of DsbA but with no oxidase activity. In this case a 3-pulse protocol was applied. e Probability of refolding of FimGSH-SH alone or with DsbA with no oxidase activity. f Force-clamp trace of FimG in the presence of both FimC and DsbA. g Percentage of disulfide bond reformation of FimG after 45 s of quenching time under different conditions. Error bars show the SD of a binomial distribution
