Fig. 6. TS structures formed during BamA folding in PE. (a and b) Two-dimensional topology diagram of BamA highlighting residues that form native-like (blue), unfolded-like (red), or non-native interactions (gray) in the transition state structure of BamA in the fast (Φ_F1-PE) (a) and slow (Φ_F2-PE) (b) pathways. Φ_F ranges used for each color code are indicated below (boxed). β-Strand numbers are annotated in (a) in blue. Residues with ΔΔG⁰_F ≤ −0.09 kcal mol⁻¹ or ≥ +0.09 kcal mol⁻¹ (ΔΔG⁰_F > 10% significance) are shown as uppercase single-letter abbreviations. Residues with ΔΔG⁰_F ≤ −0.05 kcal mol⁻¹ or ≥ +0.05 kcal mol⁻¹ (ΔΔG⁰_F between 5–9.99%) are in lowercase, italics. To minimize errors in the analyses, mutants with ΔΔG⁰_F between ± 0.0499 kcal mol⁻¹ were excluded from the topology diagram. Local zones in β-strands which show several native-like interactions are considered folded in the transition state, and are highlighted in light blue casing. Complete data in Fig. S29. (a) The Φ_F1-PE pathway shows fewer native-like interactions in the transition state, with several residues forming non-native contacts distributed across the β-strands. (b) Φ_F2-PE displays an assembled β10–β16 towards the periplasmic side of BamA. Additionally, β3–β9 are assembled towards the extracellular face of the β-barrel, suggesting the successful membrane translocation and repositioning of loops L3–L5. (c) Illustrated here are the TS structures we captured in PE. Structures (generated using PyMOL) are presented on the left, and their cartoon representations on the right extreme. PE-rich membranes (pale teal) kinetically retard rapid folding of nBamA, causing minimally structured TS ensembles. The TS ensemble is very poorly structured in the fast pathway (i) with an H-bonded β-hairpin formed only at β11–β12 (left inset), L1, T1, and T7. The TS ensemble is marginally more structured in the slow pathway (ii), with H-bonding at β5–β8 (middle inset), β10–β15 (right inset), L3–L5, and T5–T7. Both pathways involve a structural pattern of transient non-native interactions and unfolded regions formed by nBamA. Several of these interactions are also likely to occur in vivo at the membrane interface and BamA^M-bound state.