Primary structure of major outer-membrane protein I (ompF protein, porin) of Escherichia coli B/r

Abstract

The amino acid sequence of the pore-forming outer membrane protein I (porin) from Escherichia coli B/r has been determined. The polypeptide contains 340 amino acid residues resulting in a molecular weight of 37,205. The trans-membrane polypeptide has no stretches of nonpolar residues, uninterrupted by charged side chains, longer than 11 amino acid residues. Regarding polarity, the chain can be subdivided into three regions: a distinctly hydrophilic region between residues 1 and 82 (51.2% polarity), a fairly nonpolar region between residues 83 and 194 (33.9% polarity), and a more hydrophilic region up to the COOH terminus (48% polarity). These results are interpreted as evidence against a simple transmembrane structure in which the membrane is spanned by a single contiguous sequence of hydrophobic amino acids, as has been proposed, or example, for glycophorin. The cell envelope of Gram-negative bacteria possesses, in addition to the plasma membrane, an outer membrane which has perhaps more correctly also been called a porous skeletal organ (see ref. 1 for recent review) (2). This porosity is provided by proteins, the porins (e.g., refs. 3-5), which form hydrophilic channels allowing the diffusion of various low molecular weight solutes. In Escherichia coli B/r, protein 1 (6) [closely related to Rosenbusch's matrix protein from E. coli BE, (7)] is the porin responsible for the existence of these channels, which have a diameter of about 0.9 nm (8, 9). Aside from the fact that information is sparse regarding structure-function relationships of integral membrane proteins in general, the porins pose a number of interesting questions in addition to those connected with their physiological functions. In E. coli K-12 a whole family of such proteins of similar size and properties exists, and it seems that, under usual laboratory conditions, several of the corresponding structural genes are silent or nearly so (10-13). These genes are not clustered on the E. coli chromosome, and if they have arisen by duplications they might allow some insight into the evolution of this chromosome (11, 13-17). Furthermore, several of these proteins can serve as at least parts of phage receptors (18-21) and they are required for an apparent uptake of protein into the cell: mutants lacking certain such polypeptides are highly tolerant to several colicins (e.g., refs. 22 and 23). It is not known what constitutes a phage receptor area on such proteins and what is their function in colicin sensitivity. Finding answers to all these and related questions would be helped by a knowledge of the amino acid sequence of such a protein. It is also likely that the gene for the protein under study will soon become available by DNA cloning. The determination of the DNA sequence, of much interest because of the unknown control region(s), should of course also be much aided by knowledge of the primary structure of the protein. We have determined the sequence of protein I from E. coli B/r and here present its primary structure. [Strains of E. coli B/r can dif...

An unusual linkage between polysaccharides and some major proteins from the outer membrane of Escherichia coli

“…Allysine, derived from lysine by oxidative deamination, could serve to cross-link the protein to other structures containing amino groups. However, these data were not confirmed by Chen et al [20], who sequenced OmpF protein and did not find allysine residues, although these authors speculated about the possibility that modified lysine could be present in such small amounts that it escaped detection. Some molecules of OmpA protein might contain reactive modified groups.…”

An alternative topological model for Escherichia coli OmpA

“…In an attempt to correlate immunologic, biochemical, and genetic data derived from studies on proteins of the OmpA family with the three computer predictions described above, I constructed a new OmpA model. More specifically, this model was based on (1) OmpA topology data from previous studies (Chen et al, 1980;Braun & Cole, 1984;Morona et al, 1984Morona et al, , 1985Vogel & Jahnig, 1986;Freudl, 1989;Ried et al, 1994;Ruppert et al, 1994); (2) the OmpA structure predictions of Jeanteur et al,Schirmer and Cowan,and Ferenci;(3) topological information from studies on the OprF C-terminal region (Wong et al, 1993;Rawling et al, 1995); and (4) two recent reports (De Mot & Vanderleyden, 1994;Koebnik, 1995) that identified conserved residues and a common sequence motif between OmpA-related outer membrane proteins and MotB, a peptidoglycan-associated protein of gram-positive bacteria. OmpA residues involved in direct interaction with peptidoglycan are expected to lie on the periplasmic side of the membrane.…”

“…Symbols are as follows: amino acid residues in squares, sites of point mutations that result in impairment of OmpA-specific phages (Morona et al, 1984(Morona et al, , 1985; circled amino acids, residues that are conserved or homologous between OmpA-related proteins and MotB and found important for the MotB function (De Mot & Vanderleyden, 1994); +, sites of peptide insertions determined to be on the surface of the cell (Freudl, 1989;Ruppert et al, 1994); +, site of peptide insertion determined to be not exposed on the surface (Freudl et al, 1986); -, sites of peptide insertions determined to be periplasmic by in vitro proteolytic studies (Ried et al, 1994); open letter peptides, sequences homologous to monoclonal antiOprF antibody epitopes determined to be surface-exposed (Rawling et al, 1995); b, sites homologous to OprF sites proposed to be on the surface by peptide insertion studies (Rawling et al, 1995); e , cleavage site for pronase in experiments conducted with outer membranes (Chen et al, 1980); 9, sites where gaps had to be introduced for the alignment of OmpA protein sequences from five different species (Braun & Cole, 1984); -, disulfide bond; peptides in rectangles, transmembrane segments site-directed mutagenesis as important for the MotB function were placed in the periplasmically exposed loops of OmpA. (There is no evidence that all eight residues are involved in direct interactions with the peptidoglycan.…”

Gene fusions using the ompA gene coding for a major outer‐membrane protein of Escherichia coli K12

“…It is interesting to note that where larger parts of OmpA are present the fragments exhibit increased stability as shown by the presence of larger products ( M , z 31 000) encoded by pTU401 and pTU403. The OmpA protein contains two cysteine residues [3] near its C 0 2 H terminus and it is possible that these are involved in the formation of a disulfide bond, which is required for the protein to fold into a protease-resistant form. Unpublished studies from this laboratory show that if such a disulfide bond is present it is most likely of the intermolecular type.…”

“…The trans-membrane protein [2] consists of 325 amino acid residues [3] and is synthesized as a precursor, the pro-OmpA pratein, with a 21-residue signal sequence [4, 51. The protein is arranged in the outer membrane such that an NH,-terminal part, involving an area around residue 70, is exposed at the cell surface [6] and that a C0,H-terminal moiety, most likely starting around residue 177, is largely located in the periplasm…”