Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers.
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.
Arora
A
A
Department of Molecular Physiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908-0736, USA.
Rinehart
D
D
Szabo
G
G
Tamm
L K
LK
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D004553:Electric Conductivity; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008823:Micelles; D016296:Mutagenesis; D017510:Protein Folding; D014364:Tryptophan / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008823:Micelles; C078167:OMPA outer membrane proteins; D014364:Tryptophan