Assembly of the OmpF porin of Escherichia coli B

Abstract

The different conformations of the outer membrane protein OmpF of Escherichia coli B were studied with immunological probes. The antigenic determinants recognized by one monoclonal (MoF3) and two polyclonal antibodies were investigated under various conditions of solubilization which modify the association of OmpF with other membrane components, such as lipopolysaccharide. Several polymeric forms of the protein could be detected after extraction at 37°C or 56°C. The monoclonal antibody, which is specific to an exposed region of native OmpF, recognized various trimeric forms in an immunoprecipitation assay. Under the same conditions, the binding of polyclonal antibodies apparently induced strong conformational rearrangements, since the pattern of trimeric forms detected was greatly modified. The conversion of newly synthesized monomers of OmpF to the various trimer forms was investigated using these antibodies. The trimerization occurred rapidly but the appearance of the native conformation of OmpF was delayed. Some additional step was required to expose the MoF3-specific antigenic site at the surface of the trimeric form. These results are discussed in relation to the structure of OmpF and its association with lipopolysaccharide in the outer membrane.OmpC and OmpF are the major outer membrane proteins of Escherichia coli K12, functioning as passive diffusion pores for small hydrophilic molecules [l -31. These porins are assembled as trimer units at the cell surface [4 -61. The structural genes and the sequence of these proteins have been previously described and the regulation of their syntheses is being intensively studied [l, 21. In E. coli B, only one outer membrane porin, related to OmpF, is present. This protein is identical to the E. coli K12 porin except for three substitutions, at amino acid positions 66, 117 and 262 [7]. The assembly of outer membrane proteins is an intriguing and complex process requiring several steps in various cellular compartments during the export process. During and after synthesis the polypeptide chains are translocated across the cytoplasmic membrane where the cleavage of the signal sequence occurs. Several factors are required during this translocation as demonstrated by genetic and biochemical studies [8 -101. The importance of membrane fluidity, the energy requirement and the kinetics of processing are now well documented for this step.Later steps, which ensure the correct insertion of the proteins into the outer membrane, are less well understood. In the case of the outer membrane protein OmpA, an intermediate form (imp-OmpA) is detected, just after processing, at the external side of the cytoplasmic membrane [ll]. The binding of lipopolysaccharide may induce a conformational change promoting the subsequent integration into, the outer membrane. However, for OmpA neither dimeric nor trimeric assembly is required for the integration of the native protein.The maltoporin LamB [ 121 undergoes several conformational changes in order to achieve its native trimeric form in the Corr...

Rapid isolation of OmpF porin-LPS complexes suitable for structure-function studies

“…Structural investigations concomitant with functional studies employing different porin-LPS species will be useful to learn more about the porin-LPS interactions and to understand at the molecular level the actual role of each part in the regulation of the porin channel permeability. Furthermore, the different isolated porin-LPS isoforms may be an important tool to prove the hypothesis that the binding of LPS to porin, possibly corresponding to the eukaryotic glycosylation process, assists in the proper insertion of porin into the outer membrane (Pages & Bolla, 1988).…”

Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism

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Biochemistry Of Endotoxins

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