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 confirmed 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-barrel membrane 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. © 2007 Elsevier Ltd. All rights reserved. *Corresponding authorKeywords: membrane protein folding; membrane protein stability; membrane insertion; outer membrane protein; VDAC Abbreviations used: C 8 E 4 , tetraethyleneglycol-monooctylether; DDM, dodecylmaltoside; diC n:0 PC, 1, 2-diacyl-sn-glycero-3-phosphocholine (the acyl chain lengths range here from n = 10 to n = 16 carbons); diC 18:1 PC, 1, 2-dioleoyl-sn-glycero-3-phosphocholine; diPhPC, diphytanoyl phosphatidylcholine; FomA, major outer membrane protein of Fusobacterium nucleatum; hVDAC1, human isoform 1 of VDAC; IBs, inclusion bodies; LDAO, N-lauryl-N, N-dimethylamine-N-oxide; LUVs, large unilamellar vesicles; NRMSD, normalized root mean square deviation; OMP, outer membrane protein; OmpA, outer membrane protein A of E. coli; PMSF, phenyl methane sulphonyl fluoride; TSS, transformation and storage solution; VDAC, voltage dependent anion selective channel.

Folding of porin-like beta-barrel outer membrane proteins can be achieved in the presence of phospholipid vesicles, and takes place concurrently with incorporation into the membrane. The pronounced dependence found for the insertion of the protein OmpA on membrane thickness (Kleinschmidt, J. H., and L. K. Tamm. 2002. J. Mol. Biol. 324:319-330) is analyzed in terms of the effects of out-of-plane elastic fluctuations on the area dilation modulus (Evans, E., and W. Rawicz. 1990. Phys. Rev. Lett. 64:2094-2097). For unstrained large unilamellar vesicles, the elastic free energy for membrane insertion is predicted to depend on the fourth power of the membrane thickness. The influence of thermally induced bending fluctuations on the effective tilt of the OmpA beta-barrel in disaturated phosphatidylcholine membranes of different thicknesses (Ramakrishnan, M., J. Qu, C. L. Pocanschi, J. H. Kleinschmidt, and D. Marsh. 2005. Biochemistry. 44:3515-3523) is also considered. A contribution to the orientational order parameter that scales as the inverse second power of the membrane thickness is predicted.