Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli

Abstract

The arrangement of the proteins in the outer membrane of E. coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with Pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to Pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labeling with fluorescamine. The N[amino]-terminal portion of protein B was removed by Pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of a divalent cation such that Pronase digestion of the reassociated material gave protein Bp.