Characterization of the Major Envelope Protein from Escherichia coli

Abstract

The major envelope protein from Escherichia coli has been purified by differential heat extraction in dodecyl sulfate and subsequently freed of the detergent. The polypeptide is homogeneous and has a mass of 36,500 daltons. Homogeneity is based on four criteria, three of which are independent of its behavior in detergents. Its molecular weight was established by three methods independent of dodecyl sulfate binding and agrees with that derived from the mobility of the major band observed in standard dodecyl sulfate gel electrophoretic analysis of unfractionated cell envelopes after treatment at 100°. The mass of the protein is accounted for entirely, or nearly entirely, by the mass of its constituent amino acids. These results imply that dodecyl sulfate is bound in amounts corresponding to those found in most polypeptides. The protein was also isolated in association with the rigid layer of the cell by extraction of cell envelopes in 2 % dodecyl sulfate at 60°. This complex is composed of about 65 % envelope protein, the remaining mass being accounted for largely by the peptidoglycan-lipoprotein structure. In this form the protein is completely resistant to trypsin, but upon dissociation it is quickly degraded to small fragments. Unlike the dissociated polypeptide, the complexed form of the protein does not bind dodecyl sulfate tightly even upon prolonged exposure to high excess at 60°. A large fraction of the polypeptide exists as β-structure, as determined by circular dichroism and infrared spectroscopy. The 105 copies of this polypeptide per cell are arranged in a lattice structure with hexagonal symmetry and a periodicity of 7.5 nm on the outer face of the peptidoglycan. The regular array observed appears to be closely related to its quaternary structure in vivo. All strains of E. coli tested contain this protein.