TY - JOUR
T1 - Membrane topology and site‐specific mutagenesis of Pseudomonas aeruginosa porin OprD
AU - Huang, Honglin
AU - Jeanteur, Denis
AU - Pattus, Franc
AU - Hancock, Robert E.W.
N1 - Copyright:
Copyright 2016 Elsevier B.V., All rights reserved.
PY - 1995/6
Y1 - 1995/6
N2 - 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 super‐family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD‐topology model was proposed. Sixteen β‐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::Ω background. The L2‐deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.
AB - 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 super‐family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD‐topology model was proposed. Sixteen β‐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::Ω background. The L2‐deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.
UR - http://www.scopus.com/inward/record.url?scp=0029101289&partnerID=8YFLogxK
U2 - 10.1111/j.1365-2958.1995.tb02319.x
DO - 10.1111/j.1365-2958.1995.tb02319.x
M3 - Article
C2 - 7476190
AN - SCOPUS:0029101289
SN - 0950-382X
VL - 16
SP - 931
EP - 941
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 5
ER -