Pseudomonas aeruginosa porin OprF exists in two different conformations.
The major nonspecific porin of Pseudomonas aeruginosa, OprF, produces a large channel yet allows only a slow diffusion of various solutes. Here we provide an explanation of this apparent paradox. We first show, by introduction of tobacco etch virus protease cleavage site in the middle of OprF protein, that most of OprF population folds as a two-domain protein with an N-terminal beta-barrel domain and a C-terminal periplasmic domain rich in alpha-helices. However, sedimentation of unilamellar proteoliposomes through an iso-osmotic gradient showed that only about 5% of the OprF population produced open channels. Gel filtration showed that the open channel conformers tended to occur in oligomeric associations. Because the open channel conformer is likely to fold as a single domain protein with a large beta-barrel, we reasoned that residues near the C terminus may be exposed on cell surface in this conformer. Introduction of a cysteine residue at position 312 produced a functional mutant protein. By using bulky biotinylation reagents on intact cells, we showed that this cysteine residue was not exposed on cell surface in most of the OprF population. However, the minority OprF population that was biotinylated in such experiments was enriched for the conformer with pore-forming activity and had a 10-fold higher pore-forming specific activity than the bulk OprF population. Finally trypsin treatment, which preferentially cleaves the C-terminal domain of the two-domain conformer, did not affect the pore-forming activity of OprF nor did it digest the minority conformer whose residue 312 is exposed on cell surface.
Sugawara
Etsuko
E
Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA 94720-3202, USA.
Nestorovich
Ekaterina M
EM
Bezrukov
Sergey M
SM
Nikaido
Hiroshi
H
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D008968:Molecular Conformation; D018272:Porins / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D018272:Porins; C078167:OMPA outer membrane proteins; D014357:Trypsin