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J Biol Chem, Vol. 273, Issue 22, 13794-13800, May 29, 1998

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^¶, and Carmen A. Mannella^§

From the  Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, the ^§ Department of Biomedical Sciences, State University of New York, Albany, New York 12222, and the ^¶ Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

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 -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 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.

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