Fig. 2. BamA exhibits two-state cooperative folding in membrane vesicles. (a) End-state equilibrium profiles of BamA monitored using Trp fluorescence in both PC:PE and PC:PG vesicles. The 120-h folding equilibrium profiles are shown with error bars from two independent experiments. Representative unfolding profiles are also superimposed here (PC:PE-500-h; PC:PG-816-h). The mid-point of each transition (Cm) is 2.04 ± 0.05 M (PC:PE) and 2.08 ± 0.10 M (PC:PG). Errors are from five independent experiments. (b) BamA folding equilibrates in 120 h, and unfolding equilibrates in ∼20 days in PC:PE and ∼34 days in PC:PG. Shown here is the change in Cm with time for one representative folding (filled circles) and unfolding (open circles) titration. Insets: representative unfolding titrations (time course; red → green) (complete data in Fig. S5). BamA folding is a highly cooperative process with a high thermodynamic stability. Yet, BamA unfolding is slower because of the added effect of high kinetic stability. (c) Strategy for the Ala-scanning mutagenesis library of BamA generated in a Cys-less background (top) illustrated using sequences of five representative Ala variants (bottom). The positions of all mutated residues are illustrated in Fig. S7. (d) Representative equilibrium folding profiles of two intrinsically stabilizing (green squares), destabilizing (red circles), and WT-like (white diamonds) residues identified from end-state thermodynamics of BamA Xaa → Ala in both membranes. The fitted line (black) corresponds to WT BamA. Additional data in Fig. S7.
