Bacterial expression and characterization of the mitochondrial outer membrane channel: Effects of N-terminal modifications

Daniel A. Koppel, Kathleen W. Kinnally, Paul Masters, Michael Forte, Elizabeth Blachly-Dyson, Carmen A. Mannella

    Research output: Contribution to journalArticlepeer-review


    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, 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 β- sheet structure. Under the same conditions, the CD spectrum of bacterially expressed scVDAC indicates lower β-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.

    Original languageEnglish (US)
    Pages (from-to)13794-13800
    Number of pages7
    JournalJournal of Biological Chemistry
    Issue number22
    StatePublished - May 29 1998

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology


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