Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide.
We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.
Bulieris
Paula V
PV
Fachbereich Biologie, Fach M 694, Universität Konstanz, D-78457 Konstanz, Germany.
Behrens
Susanne
S
Holst
Otto
O
Kleinschmidt
Jörg H
JH
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry*; D010743:Phospholipids / Q000378:metabolism; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008070:Lipopolysaccharides; D018832:Molecular Chaperones; D010743:Phospholipids; C072721:Skp protein, E coli; C078167:OMPA outer membrane proteins