title: Bacterial expression, purification and characterization of a rice voltage-dependent, anion-selective channel isoform, OsVDAC4.
abstract: The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and forms the major conduit for metabolite transport across this membrane. VDACs from different sources show varied primary sequence but conserved functional properties. Here, we report on the characterization of a rice channel, OsVDAC4, which complements a VDAC1 deficiency in yeast. We present a consensus secondary structure prediction of an N-terminal α-helix and 19 β-strands. Bacterially expressed OsVDAC4 was purified from inclusion bodies into detergent-containing solution, where it is largely helical. Detergent-solubilized OsVDAC4 inserts spontaneously into artificial membranes of two topologies-spherical liposomes and planar bilayers. Insertion into liposomes results in an increase in β-structure. Transport of polyethylene glycols was used to estimate a pore diameter of ~2.6 nm in liposomes. Channels formed in planar bilayers exhibit large conductance (4.6 ± 0.3 nS in 1 M KCl), strong voltage dependence and weak anion selectivity. The open state of the channel is shown to be permeable to ATP. These data are consistent with a large β-barrel pore formed by OsVDAC4 on inserting into membranes. This study forms a platform to carry out studies of the interaction of OsVDAC4 with putative modulators.
authors: National Centre for Biological Sciences, TIFR, UAS-GKVK Campus, Bangalore, India.
authors: Ashwini
authors: A
authors: Godbole
authors: Rohan
authors: R
authors: Mitra
authors: Ashvini K
authors: AK
authors: Dubey
authors: Palakolanu S
authors: PS
authors: Reddy
authors: M K
authors: MK
authors: Mathew
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology*; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D050995:Voltage-Dependent Anion Channel 1 / Q000737:chemistry / Q000235:genetics; D050994:Voltage-Dependent Anion Channels / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000838:Anions; D003902:Detergents; D008051:Lipid Bilayers; D008081:Liposomes; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D050994:Voltage-Dependent Anion Channels; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D050995:Voltage-Dependent Anion Channel 1