Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded β-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 proteinsin 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.
