Abstract
Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-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.
Highlights
The outer membrane of Gram-negative bacteria serves as a molecular sieve, resisting the entry of noxious compounds, while at the same time allowing the uptake of essential nutrients
The crystal structure of the N-terminal domain of Outer membrane protein A (OmpA), which was obtained from crystals formed from an OmpA fragment that was solubilized in the detergent C8E4, indicated no obvious aqueous pore, as the water-filled cavities were not connected in that structure [7]
In this experiment(s) OmpA in C8E4 micelles was added to the cis compartment next to DPhPC/n-decane bilayers and equilibrated, and a 100-mV potential was applied to the trans compartment
Summary
Outer membrane protein; DPhPC, diphytanoylphosphatidylcholine; pS, picosiemens; LamB, maltophorin; Fhu, ferric hydroxamate uptake receptor; Fep, ferric enterobactin receptor. Providing structural stability to the cell appears to be one of the main functions of OmpA This is probably accomplished by linking through the C-terminal domain the outer membrane to the periplasmic peptidoglycan. Because the formation of ion channels by OmpA has not been universally accepted and to develop a practical assay for the refolding of OmpA, we re-investigated the channel activity of OmpA in a well defined reconstituted lipid bilayer system We found that both native OmpA and OmpA that was refolded in the detergent C8E4 forms two types of ion-conducting channels in planar bilayer membranes. The smaller but not the larger conductance state was observed when the N-terminal transmembrane domain, i.e. a fragment comprising residues 1–176, was refolded and incorporated into planar lipid bilayers.
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