BACTERIAL EXPRESSION AND CHARACTERIZATION OF THE MITOCHONDRIAL OUTER-MEMBRANE CHANNEL - EFFECTS OF N-TERMINAL MODIFICATIONS

Several forms of the voltage-dependent anion-selective channel (VDAC) have been expressed at high yield in Escherichia coli, Full length con structs of the proteins of Neurospora crassa and Saccharomyces cerevis iae (ncVDAC and scVDAC) have been made with 20-residue-long, thrombin- cleavable, His(6)-containing N-terminal extensions, ncVDAC purified fr om bacteria or mitochondria displays a far-UV CD spectrum (in 1% laury l dimethylamine oxide at pH 6-8) similar to that of bacterial porins, indicating extensive beta-sheet structure, Under the same conditions, the CD spectrum of bacterially expressed scVDAC indicates lower beta-s heet content, albeit higher than that of mitochondrial scVDAC under th e same conditions. In phospholipid bilayers, the bacterially expressed proteins (with or without N-terminal extensions) form typical VDAC-li ke channels with stable, large conductance open states (4-4.5 nanosiem ens in 1 m KCI) and voltage-dependent transitions to a predominant sub state (about 2 nanosiemens). A variant of scVDAC missing the first eig ht residues and having no N-terminal extension also has been expressed in E, coli, The truncated protein has a CD spectrum similar to that o f mitochondrial scVDAC, but its channel activity is abnormal, exhibiti ng an unstable open state and rapid transitions between multiple subco nductance levels.