Abstract
Using ion channel reconstitution in planar lipid bilayers, we examined the channel-forming activity of subfractions of Pseudomonas aeruginosa OprF, which was shown to exist in two different conformations: a minority single domain conformer and a majority two-domain conformer (Sugawara, E., Nestorovich, E. M., Bezrukov, S. M., and Nikaido, H. (2006) J. Biol. Chem. 281, 16220-16229). With the fraction depleted for the single domain conformer, we were unable to detect formation of any channels with well defined conductance levels. With the unfractionated OprF, we saw only rare channel formation. However, with the single domain-enriched fraction of OprF, we observed regular insertion of channels with highly reproducible conductances. Single OprF channels demonstrate rich kinetic behavior exhibiting spontaneous transitions between several subconformations that differ in ionic conductance and radius measured in polymer exclusion experiments. Although we showed that the effective radius of the most conductive conformation exceeds that of the general outer membrane porin of Escherichia coli, OmpF, we also found that a single OprF channel mainly exists in weakly conductive subconformations and switches to the fully open state for a short time only. Therefore, the low permeability of OprF reported earlier may be due to two factors: mainly to the paucity of the single domain conformer in the OprF population and secondly to the predominance of weakly conductive subconformations within the single domain conformer.
Highlights
Using ion channel reconstitution in planar lipid bilayers, we examined the channel-forming activity of subfractions of Pseudomonas aeruginosa OprF, which was shown to exist in two different conformations: a minority single domain conformer and a majority two-domain conformer
We showed that the effective radius of the most conductive conformation exceeds that of the general outer membrane porin of Escherichia coli, OmpF, we found that a single OprF channel mainly exists in weakly conductive subconformations and switches to the fully open state for a short time only
The protein coded by the truncated oprF gene was preceded by a hexahistidine tag and the signal sequence of OmpA and under our conditions nearly exclusively found in the outer membrane fraction
Summary
Using ion channel reconstitution in planar lipid bilayers, we examined the channel-forming activity of subfractions of Pseudomonas aeruginosa OprF, which was shown to exist in two different conformations: a minority single domain conformer and a majority two-domain conformer Kinetic studies of solute diffusion [3, 4], using osmotic swelling of proteoliposomes, have shown that OprF has much lower permeability (i.e. allows much slower permeation of the same test solute) than the classical trimeric porins of E. coli but forms channels that are wider than the channels of E. coli porins because the diffusion rates are much less influenced by the size of the oligosaccharide solutes. Intact E. coli cells expressing OprF porin from plasmid-coded gene are capable of growing on raffinose [5], which is too large (505 daltons) to serve as an effective carbon source for the wild-type E. coli
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