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 journalArticle

Abstract

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
Volume273
Issue number22
DOIs
StatePublished - May 29 1998

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Voltage-Dependent Anion Channels
Ion Channels
Escherichia coli
Porins
Neurospora crassa
Proteins
Mitochondria
Yeast
Oxides
Saccharomyces cerevisiae
Phospholipids
Bacteria
Electric potential

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bacterial expression and characterization of the mitochondrial outer membrane channel : Effects of N-terminal modifications. / Koppel, Daniel A.; Kinnally, Kathleen W.; Masters, Paul; Forte, Michael; Blachly-Dyson, Elizabeth; Mannella, Carmen A.

In: Journal of Biological Chemistry, Vol. 273, No. 22, 29.05.1998, p. 13794-13800.

Research output: Contribution to journalArticle

Koppel, Daniel A. ; Kinnally, Kathleen W. ; Masters, Paul ; Forte, Michael ; Blachly-Dyson, Elizabeth ; Mannella, Carmen A. / Bacterial expression and characterization of the mitochondrial outer membrane channel : Effects of N-terminal modifications. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 22. pp. 13794-13800.
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