Factors affecting the folding of Pseudomonas aeruginosa OprF porin into the one-domain open conformer.
Pseudomonas aeruginosa OprF is a largely monomeric outer membrane protein that allows the slow, nonspecific transmembrane diffusion of solutes. This protein folds into two different conformers, with the majority conformer folding into a two-domain conformation that has no porin activity and the minority conformer into a one-domain conformation with high porin activity and presumably consisting of a large β barrel. We examined the factors that control the divergent folding pathways of OprF. OprF contains four Cys residues in the linker region connecting the N-terminal β-barrel domain and the C-terminal globular domain in the majority conformer. Prevention of disulfide bond formation either by expression of OprF in an Escherichia coli dsbA strain grown with dithiothreitol or by replacement of all Cys residues with serine (CS OprF) increased the specific pore-forming activity of OprF significantly. Replacement of Phe160 with Ile at the end of the β-barrel termination signal as well as replacement of Lys164 in the linker region with Gly, Cys, or Glu increased porin activity 2-fold. Improving a potential β-barrel termination signal in the periplasmic domain by replacement of Asp211 with asparagine also increased porin activity. The porin activity could be improved about 5-fold by the combination of these replacements. OprF mutants with higher porin activity were shown to contain more one-domain conformers by surface labeling of the A312C residue in intact cells, as this residue is located in the periplasmic domain in the two-domain conformers. Finally, when the OprF protein was expressed in an E. coli strain lacking the periplasmic chaperone Skp, the CS OprF protein exhibited increased pore-forming activity.
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
Etsuko
E
Sugawara
Keiji
K
Nagano
Hiroshi
H
Nikaido
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C547605:OprF protein, Pseudomonas aeruginosa