Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Abstract

Spontaneous membrane insertion and folding of β-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 α-helical or β-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane β-barrel with an additional N-terminal α-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 confirme folding of hVDAC1 to its active form. hVDAC1 developed first β-sheet secondary structure in aqueous solution, while the α-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial β-barrelmembrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of β-sheet and lower content of α-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of β-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial β-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 β-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.