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J. Biol. Chem., Vol. 281, Issue 24, 16220-16229, June 16, 2006

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Pseudomonas aeruginosa Porin OprF Exists in Two Different Conformations^*

Etsuko Sugawara, Ekaterina M. Nestorovich, Sergey M. Bezrukov, and Hiroshi Nikaido¹

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

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 -barrel domain and a C-terminal periplasmic domain rich in -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 -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.

Received for publication, January 23, 2006 , and in revised form, March 31, 2006.

^* This work was supported by National Institutes of Health Grant AI-09644 (to H. N.). 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: Dept. of Molecular and Cell Biology, 426 Barker Hall, University of California, Berkeley, CA 94720-3202. Tel.: 510-642-2027; Fax: 510-643-6334; E-mail: nhiroshi{at}berkeley.edu.

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