Bacterial Expression, Purification and Characterization of a Rice Voltage-Dependent, Anion-Selective Channel Isoform, OsVDAC4

Bacterial Expression, Purification and Characterization of a Rice Voltage-Dependent,... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Bacterial Expression, Purification and Characterization of a Rice Voltage-Dependent, Anion-Selective Channel Isoform, OsVDAC4

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Publisher
Springer-Verlag
Copyright
Copyright © 2011 by Springer Science+Business Media, LLC
Subject
Life Sciences; Human Physiology; Biochemistry, general
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-011-9399-x
Publisher site
See Article on Publisher Site

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