Effects of the Periplasmic Domain of BamA, the Essential Transmembrane Protein of the Beta-Barrel Assembly Machinery (BAM), in Folding and Insertion of the Outer Membrane Protein A

Abstract

In Escherichia coli, folding and insertion of outer membrane proteins (OMPs) is facilitated by the β-barrel assembly machinery (BAM), composed of the integral protein BamA and the four lipoproteins BamB, BamC, BamA and BamE. BamA consists of a transmembrane β-barrel and a soluble periplasmic domain (PD-BamA), comprised of five polypeptide transport-associated (POTRA) domains. Although it is known that PD-BamA is necessary for the activity of the BAM-complex, its mechanism is not understood. To examine whether PD-BamA promotes folding and insertion of OMPs into lipid membranes, we expressed and isolated it from E. coli. The secondary structure of PD-BamA was determined by CD spectroscopy to 36% α-helix, 15% β-strand, 20% β-turns, and 29% random coil. For OMP folding experiments, we isolated outer membrane protein A (OmpA) from E. coli in unfolded form in urea-solution. To investigate the effect of PD-BamA on the kinetics of OmpA folding into the model membranes different sets of experiments were performed. In parallel experiments, in the absence and presence of PD-BamA, the dependence of OmpA folding was examined by gel electrophoresis as described [1]. The effects of temperature, lipid composition, and lipid concentration on PD-BamA function were investigated. PD-BamA was found to promote folding of OmpA into lipid bilayers when phosphatidylglycerol (PG) was present in the membrane. No effect on the folding of OmpA could be observed in the absence of PG. Fluorescence spectroscopy showed that PD-BamA binds to lipid bilayers, but only when these contain PG. The kinetics of OmpA folding, determined at various PD-BamA/OmpA ratios, indicated a 1:1 stoichiometry of the interaction of PD-BamA and unfolded OmpA.[1] Kleinschmidt, J.H. (2003) Cell. Mol. Life Sci. 60, 1547-1558.