Several forms of the voltage-dependent anion-selective channel (VDAC) have been expressed at high yield in Escherichia coli. Full-length constructs of the proteins of Neurospora crassa and Saccharomyces cerevisiae (ncVDAC and scVDAC) have been made with 20-residue-long, thrombincleavable, His 6 -containing N-terminal extensions. ncVDAC purified from bacteria or mitochondria displays a far-UV CD spectrum (in 1% lauryl dimethylamine oxide at pH 6-8) similar to that of bacterial porins, indicating extensive ␤-sheet structure. Under the same conditions, the CD spectrum of bacterially expressed scVDAC indicates lower ␤-sheet content, albeit higher than that of mitochondrial scVDAC under the same conditions. In phospholipid bilayers, the bacterially expressed proteins (with or without Nterminal extensions) form typical VDAC-like channels with stable, large conductance open states (4-4.5 nanosiemens in 1 M KCl) and voltage-dependent transitions to a predominant substate (about 2 nanosiemens). A variant of scVDAC missing the first eight residues and having no N-terminal extension also has been expressed in E. coli. The truncated protein has a CD spectrum similar to that of mitochondrial scVDAC, but its channel activity is abnormal, exhibiting an unstable open state and rapid transitions between multiple subconductance levels. VDAC1 or mitochondrial porin is the most abundant transport protein in the mitochondrial outer membrane. Although VDAC proteins from different species have only weak sequence homology, their functional properties are highly conserved (1, 2). In artificial lipid bilayers, the channel occupies a high conductance, anion-selective open state (4 -4.5 nS in 1 M KCl) at small membrane potentials and switches, at potential amplitudes above 30 -40 mV, to lower conductance substates (2-2.5 nS) that are cation-selective and less permeable to ATP and ADP (3-5). The critical potential at which gating occurs is decreased in the presence of polyanions and a soluble mitochondrial protein fraction (6, 7). VDAC sequences from a number of species contain numerous stretches of alternating hydrophobic and hydrophilic residues (e.g. see Refs. 8 and 9), suggesting an amphipathic ␤-barrel motif like that of the bacterial porins (10, 11). Analysis of VDAC sequences with the Gibbs sampler indicates the presence of numerous matches to a residue-frequency motif associated with transmembrane ␤-strands in bacterial porins (12). Circular dichroism studies of VDAC purified from Neurospora crassa in both lipids and nondenaturing detergents (13) suggest that ncVDAC has a high ␤-sheet content, consistent with a bacterial porin-like ␤-barrel structure (e.g. see Ref. 14).The main focus of our research is to determine the molecular basis for VDAC's functional properties, in particular, its permeability and mechanism of gating. In this report, we describe the bacterial expression of functional VDAC from Saccharomyces cerevisiae and N. crassa and compare the effects of Nterminal extension and truncation on the properties of this channel ...

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