MEMBRANE TOPOLOGY AND SITE-SPECIFIC MUTAGENESIS OF PSEUDOMONAS-AERUGINOSA PORIN OPRD

Pseudomonas aeruginosa OprD is a 420-amino-acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non-specific porin superfamily, Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD-topology model was proposed, Sixteen P-strands were predicted, connected by short loops at the periplasmic side, The eight external loops were of variable length but tended to be much longer than the periplasmic ones, Polymerase chain reaction (PCR)-based site-specific mutagenesis was performed to delete separately short stretches (4-8 amino acid residues) from each of the predicted external loops, The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD::Omega background, The La-deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.