Isolation and preliminary characterization of wild-type OmpC porin dimers from Escherichia coli K-12.
Escherichia coli K-12 strain PLB3255 contains a mutation in the ompF gene that results in a 15 amino acid deletion in the porin protein. The mutation (dex) appears to increase the OmpF channel size, allowing the PLB3255 cells to grow on maltodextrins in the absence of a functional maltoporin. Porin isolated from strain PLB3255, which contains a wild-type ompC gene, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shown to contain 50-60% trimer aggregates and 35-40% of a 50-kDa "dimer". When the porin isolate was heated to 100 degrees C and separated on SDS gels containing 8 M urea, both the trimer and the "dimer" were recovered in a single band at 36 kDa that corresponded in mobility to wild-type OmpC porin. An analysis of the temperature stability of the isolate showed that the OmpC "dimer" was less stable and denatured at 66 degrees C compared to 81 degrees C for the trimer. To separate these aggregates, the unheated porin was suspended in 30% SDS, applied to a Sephadex G-200 gel filtration column, and eluted with 0.5% sodium deoxycholate. Two peaks were recovered containing separated trimers and "dimers". Circular dichroism spectra of isolated dimers and trimers indicated similar amounts of beta-structure. The isolated dimers and trimers were reconstituted into artificial membranes. Electrical conductance across planar bilayer lipid membranes and liposome swelling assays showed that the two isolates had similar channel-forming activity. Four other ompF deletion mutants of the same phenotype were also shown to produce 50-kDa OmpC porin "dimers".(ABSTRACT TRUNCATED AT 250 WORDS)
Rocque
W J
WJ
Department of Biochemistry, Michigan State University, East Lansing 48824.
McGroarty
E J
EJ
D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000032:analysis* / Q000254:growth & development / Q000378:metabolism; D007473:Ion Channels / Q000032:analysis* / Q000378:metabolism; D008051:Lipid Bilayers; D008081:Liposomes; D046911:Macromolecular Substances; D008970:Molecular Weight; D018272:Porins
D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D046911:Macromolecular Substances; D018272:Porins