Properties and organization of the proteins in the outer membrane of Escherichia coli

Two major proteins of the outer membrane of Escherichia coli, the matrix protein, A (M.W. 36,500) and the heat-modifiable protein, B were purified and partially characterized. Both have a low content of cysteine, . an excess of acidic amino acids over basic and a moderate content of hydrophobic amino acids. Protein B (M.W. 28,500) was converted to form B* (M.W. 33j^00) upon heating in the presence of sodium dodecyl sulfate at temperatures higher than 50°C. Physical studies showed that protein B unfolds upon heating without a large increase in binding of sodium dodecyl sulfate. It is proposed that protein B as extracted from the membrane contains some native structure which is lost upon heating. The level of protein A1, a major outer membrane protein in glucose-grown cells, was decreased in cells grown on other carbon sources with a concomitant increase in the amount of protein A2. Both proteins were tightly associated with the peptidoglycan and had similar amino acid composition, suggesting that they play the same role in the outer membrane. The organization of proteins in the outer membrane of E. coli was studied by proteolytic digestion, covalent labelling and crosslinking. The proteins of the outer membrane were inaccessible to pronase in intact cells and the cells altered in their lipopolysaccharide component. The protein components of isolated outer membrane preparations varied in their rates of digestion and labelling with fluoresca- ' mine, suggesting that they are asymmetrically arranged in the membrane. The proteins most rapidly degraded (proteins B,C-,D1 and E) were judged to be exposed at the surface of the membrane, while those resistant to digestion (proteins A1,A2 and D2) must be protected by their arrangement in the membrane. Digestion of outer membrane preparations with pronase left a fragment derived from protein B (protein Bp) embedded in the membrane. This fragment was not enriched in hydrophobic amino acids relative to protein B. Protein B could be reassociated with itself j without phospholipid or lipopolysacchari.de such that pronase digestion of the reassociated material gave protein Bp. These results suggest that protein B may not be held in the membrane primarily by hydrophobic interactions. The resistance of proteins A1 and A2 to protease digestion is likely due to protein-protein interactions since oligomers of protein A could be isolated. Treatment of protein A1- or A2-peptidogly-can complexes with dithiobis (succinimidyl propionate) or glutaraldehyde produced dimer, trimer and higher oligomers of protein A. No crosslinking of protein A to the peptidoglycan was detected. The proteins of the isolated outer membrane varied in their ease of crosslinking. Protein B, but not the pronase-resistant fragment, protein Bp, was readily crosslinked to give high molecular weight oligomers, while protein A formed dimers and trimers under the same conditions. No crosslinking of protein A to B was detected. Crosslinking of cell wall preparations showed that protein B and the free form of the lipoprotein, F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. These results suggest that specific protein-protein interactions occur in the outer membrane. A model for the arrangement of the proteins in the outer membrane of E. coli, summarizing the results of proteolytic digestion, covalent labelling and crosslinking, is presented.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat