Fig. 5. BamA unfolding is biphasic and proceeds through parallel pathways in PC:PE and PC:PG. (a) Representative folding (k_f1, k_f2, k_f3, and k_f4) and unfolding (k_u1 and k_u2) kinetics of BamA monitored using changes in the total fluorescence emission of the 13 intrinsic Trp residues, provide two chevron profiles (fit lines) in both membranes. Each unfolding rate links with two of the folding rates (k_f1–k_u1 and k_f3–k_u2; fits shown as solid lines), displaying amplitude dependence (see Fig. S22 and S23), and providing the change in kinetic free energy. The folding rates k_f2 and k_f4 may correspond to off-pathway events or on-pathway TS structures. At values closer to the C_m, the process is too slow for accurately measuring the rate constants (also see Fig. S22). Interrupted folding (double jump re-unfolding, DJ_RUF) measurements (●) display two rates that are in good agreement with k_u1 and k_u2 (shown here are rates for t_age = 999 s; rates from other t_age in Fig. S25), and support BamA (un)folding via parallel pathways. (b–d) Unfolding rate constants (k_u values) for WT BamA (b) and its mutants were obtained from fits of the unfolding rates to a linear equation (fits shown as solid lines) using shared m-values (listed in Table S2). (c and d) Unfolding data for three representative BamA variants that are folded (left, blue), unfolded (middle, red), or form non-native interactions (right, gray) in the transition state (TS) structure, in PC:PE (c) and PC:PG (d). The positions of these residues are indicated (right extreme) on the structure of BamA. See Tables S10–S13 for the measured kinetic parameters.