Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida.
We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.
D000333:Aeromonas / Q000032:analysis*; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D004553:Electric Conductivity; D007473:Ion Channels / Q000032:analysis*; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D008970:Molecular Weight; D018272:Porins; D012965:Sodium Chloride / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D018272:Porins; D012965:Sodium Chloride