title: In vitro association of fragments of a beta-sheet membrane protein.
abstract: Although the beta-barrel membrane protein OmpA can be produced in a biologically active form in E. coli from co-expressed fragments, the fragments have not been demonstrated to associate in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex according to the SDS band-shift assay. We are able to convert 25-35% of the fragment populations to non-covalent but SDS-stable complexes. The periplasmic chaperone Skp effectively prevented this association. Two separately expressed and purified overlapping fragments of OmpA can form a protease-resistant complex that undergoes the characteristic band-shift upon heating. Our work demonstrates that although membrane insertion and folding of beta-barrel membrane proteins may be a cooperative process, the fragments can associate in vitro without any additional components. However, the low yield and slow folding rates indicate that partially unfolded or destabilized beta-sheet membrane proteins can potentially engage in many non-native interactions.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors.forename: D
authors.initials: D
authors.lastname: Debnath
authors.forename: K L
authors.initials: KL
authors.lastname: Nielsen
authors.forename: D E
authors.initials: DE
authors.lastname: Otzen
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C072721:Skp protein, E coli; C078167:OMPA outer membrane proteins; D010447:Peptide Hydrolases