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J. Biol. Chem., Vol. 281, Issue 24, 16230-16237, June 16, 2006

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Pseudomonas aeruginosa Porin OprF

PROPERTIES OF THE CHANNEL^*

Ekaterina M. Nestorovich, Etsuko Sugawara, Hiroshi Nikaido, and Sergey M. Bezrukov¹

From the Laboratory of Physical and Structural Biology, NICHD, National Institutes of Health, Bethesda, Maryland 20892-0924 and the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

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.

Received for publication, January 23, 2006 , and in revised form, April 13, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratory of Physical and Structural Biology, NICHD, National Institutes of Health, Bldg. 9 Rm. 1N124, 9 Memorial Dr., Bethesda, MD 20892-0924. Tel.: 301-402-4701; Fax: 301-496-2172; E-mail: bezrukos{at}mail.nih.gov.

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