Discovery and Characterization of Two Folded Intermediates for Outer Membrane Protein TolC Biogenesis

[Display omitted] •TolC is the outer membrane protein responsible for antibiotic efflux in E. coli.•TolC folding involves the formation of two folded intermediates.•The first intermediate is a mostly helical monomer.•The second intermediate, a trimer, forms regardless of the presence of lipid.•These folding intermediates provide possible targets for antibiotic efflux inhibitors. TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.