Resolving the Native Structure of Escherichia Coli OmpA

Abstract

Modification of the sorting signal (residues 163-169) of outer membrane protein A (OmpA) of Escherichia coli by amphiphilic, water-insoluble oligo-(R)-3-hydroxybutyrates (cOHB) enables it to form narrow pores of ∼ 80 pS in planar lipid bilayers at room temperature. Here we show that additional modifications of the C-terminal domain of OmpA in the periplasm enable it to refold into large pores in the outer membranes. Both narrow and large pore conformers migrate as 30 kDa proteins on SDS-PAGE gels. OmpA isolated from outer membranes (M-OmpA) refolds into large pores of ∼ 450 pS after incubation in micelles or planar bilayers at elevated temperatures (Ea = 33.2 kcal/mol), whereas OmpA isolated from cytoplasmic inclusion bodies (I-OmpA) treated in the same manner continues to form only narrow pores. Western blot immunoassay using anti-OHB IgG and1H-NMR indicate that chymotrypsin-generated C-terminal segment 264-325 of M-OmpA contains cOHB, whereas the same segment of I-OmpA does not. Importantly, the narrow to large pore transition also fails to occur when M-OmpA is exposed to disulfide bond reducing agents. The results indicate that cOHB-modification of the sorting signal in the cytoplasm and of the C-terminal segment 264-325 in the periplasm as well as C290 -C302 disulfide bond formation in the periplasm are all necessary steps in folding OmpA to its native large pore structure. They further suggest that cOHB modification may be an important factor in protein targeting and protein folding.