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.
Koppel
D A
DA
Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.
Kinnally
K W
KW
Masters
P
P
Forte
M
M
Blachly-Dyson
E
E
Mannella
C A
CA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003902:Detergents; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000378:metabolism; D018272:Porins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D050994:Voltage-Dependent Anion Channels
D003902:Detergents; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D018272:Porins; D050994:Voltage-Dependent Anion Channels; D012967:Sodium Dodecyl Sulfate