The Peripheral Outer Membrane Protein BamB from E. coli Binds in a Random Surface Orientation to Lipid Membranes. a Site-Directed Fluorescence Study

Abstract

The β-barrel assembly machinery (BAM) complex of Gram-negative bacteria like E. coli is required for folding and insertion of outer membrane proteins (OMPs) into the outer membrane. In E. coli, the complex consists of the subunits BamA, B, C, D, and E. While BamA contains a transmembrane domain, the other subunits are peripheral membrane proteins. Deletion of bamB leads to reduced concentrations of folded OMPs, like OmpA or LamB. Here we have investigated the interface of BamB with lipid bilayers. Amino acid residues distributed over the entire surface of BamB were selected for replacement by a cysteine to prepare a range of single-cysteine mutants of BamB. Correct structure of the isolated mutants was confirmed by circular dichroism spectroscopy and mutants were labeled with fluorescent dye IANBD at the cysteine. Fluorescence spectroscopy and fluorescence quenching by spin-labeled lipids demonstrated that the entire surface of BamB interacts with the lipid bilayer, indicating a random surface orientation of BamB. The N-terminal lipid modification of BamB was not required for membrane binding.An increased binding of BamB was observed for membranes composed of PG only, likely because of electrostatic interactions. CD spectroscopy indicated that PG alters the structure of BamB, converting about ∼8 % of the residues that form β-strands to α-helical conformation. These observations may have implications for the function of BamB to improve folding of OMPs into the outer membrane.