title: Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer.
abstract: Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.
authors.affiliation: Fachbereich Biologie, Universität Konstanz, Universitätsstrasse 10, D-78464 Konstanz, Germany.
authors.forename: Baladhandapani
authors.initials: B
authors.lastname: Shanmugavadivu
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Apell
authors.forename: Thomas
authors.initials: T
authors.lastname: Meins
authors.forename: Kornelius
authors.initials: K
authors.lastname: Zeth
authors.forename: Jörg H
authors.initials: JH
authors.lastname: Kleinschmidt
mesh_terms: D003902:Detergents / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008823:Micelles; D008954:Models, Biological; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D003902:Detergents; D008051:Lipid Bilayers; D008823:Micelles; D010446:Peptide Fragments; C494762:VDAC1 protein, human; D012967:Sodium Dodecyl Sulfate; D050995:Voltage-Dependent Anion Channel 1