Fig. 1. Assembly of BamA in host–guest membranes and validation of folding. (a) Cartoon representation of a typical E. coli cell (left), illustrating the BAM complex in its OM (middle). BAM facilitates energy-independent insertion of nOMPs. BamA, with its N-terminal POTRA domain and C-terminal 16-stranded transmembrane β-barrel (left), along with the four lipoproteins BamB-E, together constitute the BAM complex. Created using Biorender.com and PyMOL. (b) The 13 Trp residues of BamA (PDB ID: 5AYW) are highlighted on the protein structure. Also indicated are the positions of the two Cys → Ser substitutions on loop 6 we carried out to obtain Cys-less BamA. (c) Chemical structures of the 14-C lipids used in this study. (d) BamA folding in LUV membranes (borate buffer, pH 10.0) by rapid dilution of the unfolded protein to achieve an LPR of 1042:1 results in unidirectional BamA assembly (schematic created using Biorender.com) (also see Table S1). (e) Folded BamA displays an electrophoretic mobility shift distinct from the unfolded protein in cold Tricine-SDS-PAGE, and (f) resistance to pulse proteolysis by proteinase K (PK), in both PE- and PG-containing membranes (also see Fig. S3 and S4). As seen with other bacterial OMPs,^51,77 BamA exhibits faster electrophoretic mobility upon folding. The membrane protects the β-barrel from proteolysis, while the N-terminal extramembranous POTRA domain, albeit folded, undergoes near-complete proteolytic digestion.⁴⁸ The latter observation confirms a unidirectional insertion of BamA in the vesicle, with the extramembranous loops positioned within the vesicle lumen and the water-soluble POTRA oriented outside the vesicle (as illustrated in 1d). (g) Folded BamA displays a far-UV circular dichroism spectrum characteristic of a β-rich protein upon folding in both PC:PE (left, teal) and PC:PG (right, purple) membranes, with molar ellipticity (ME) values of −6.26 × 10⁶ deg cm² dmol⁻¹ (PC:PE) and −6.50 × 10⁶ deg cm² dmol⁻¹ (PC:PG) at 220 nm. TEM images of the LUVs are shown as an inset (top left). An increase in Trp fluorescence emission, accompanied by a blue-shifted λ_em-max is also observed upon BamA folding (inset, bottom right; folded: filled circles, unfolded: open circles).