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 constructs of the proteins of Neurospora crassa and Saccharomyces cerevisiae (ncVDAC and scVDAC) have been made with 20-residue-long, thrombin-cleavable, His6-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 beta-sheet structure. Under the same conditions, the CD spectrum of bacterially expressed scVDAC indicates lower beta-sheet content, albeit higher than that of mitochondrial scVDAC under the same conditions. In phospholipid bilayers, the bacterially expressed proteins (with or without N-terminal 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.