Fig. 5: Effect of ligand binding on co-translocational unfolding and refolding.

a A two-histidine mutant was designed to replace a salt-bridge between Asp-48 and Lys-100 in Trx. The salt bridge present in the native state (N*) is broken in the intermediate (I*)²⁸. This mutant fluctuated between the N* and I* states at +40 mV (gray, raw data; black, signal filtered at 1 kHz). Right: dependence of the refolding kinetics on the applied potential. The two-histidine mutant (red) showed an ~2-fold decrease in the refolding rate compared to Trx-V5-C109 (black). b When 5 mM Ni²⁺ was added, the two histidine side-chains coordinated the cation altering the kinetics (+40 mV: gray, raw data; black, signal filtered at 1 kHz). Right: dependence of the refolding kinetics on the applied potential. In the presence of 5 mM Ni²⁺, the refolding rate (green) increased ~5-fold, demonstrating an interaction between residues 48 and 100 in the transition state for refolding of the intermediate (I*). The straight lines in a and b all have the same slope (obtained by a global best-fit to first order polynomials).