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J Biol Chem, Vol. 275, Issue 3, 1594-1600, January 21, 2000
From the Department of Molecular Physiology and Biological Physics
and Center for Structural Biology, University of Virginia Health
Sciences Center, Charlottesville, Virginia 22908-0736
Outer membrane protein A (OmpA), a major
structural protein of the outer membrane of Escherichia
coli, consists of an N-terminal 8-stranded
Refolded Outer Membrane Protein A of Escherichia coli
Forms Ion Channels with Two Conductance States in Planar Lipid
Bilayers*
-barrel
transmembrane domain and a C-terminal periplasmic domain. OmpA has
served as an excellent model for studying the mechanism of insertion,
folding, and assembly of constitutive integral membrane proteins
in vivo and in vitro. The function of OmpA is
currently not well understood. Particularly, the question whether or
not OmpA forms an ion channel and/or nonspecific pore for uncharged
larger solutes, as some other porins do, has been controversial. We
have incorporated detergent-purified OmpA into planar lipid bilayers
and studied its permeability to ions by single channel conductance
measurements. In 1 M KCl, OmpA formed small (50-80 pS) and
large (260-320 pS) channels. These two conductance states were
interconvertible, presumably corresponding to two different
conformations of OmpA in the membrane. The smaller channels are
associated with the N-terminal transmembrane domain, whereas both
domains are required to form the larger channels. The two channel
activities provide a new functional assay for the refolding in
vitro of the two respective domains of OmpA. Wild-type and five
single tryptophan mutants of urea-denatured OmpA are shown to refold
into functional channels in lipid bilayers.
*
This work was supported by Grants GM51329 (to L. K. T.)
and HL37127 (to G. S.) from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Physiology and Biological Physics, University of Virginia Health Sciences Cntr., P.O. Box 800736, Charlottesville, VA 22908-0736. Tel.:
804-982-3578; Fax: 804-982-1616; E-mail: lkt2e@virginia.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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