TolC of Escherichia coli functions as an outer membrane channel.
Reconstitution experiments were performed with TolC from Escherichia coli outer membrane by using the lipid bilayer membrane technique. TolC was purified by elution of the oligomeric and the monomeric forms out of preparative SDS-PAGE. The oligomeric but not the monomeric form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes. Investigation of the membrane activity in single-channel experiments suggested that TolC formed ion-permeable channels. The channels of 80 pS in 1 M KCl had a much smaller single-channel conductance than the general diffusion pores of E. coli outer membrane (1500 pS). The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration which indicated either ion binding or charge effects. Titration of TolC-induced membrane conductance with peptides lead to a dose-dependent decrease of the conductance. This result suggested that TolC contained a binding site for peptides. A dissociation constant of 20 mM was calculated for the binding of the tripeptide H-Gly-Gly-Leu-OH to the binding site. The results are consistent with the assumption that TolC acts as an outer membrane channel for peptides.
Benz
R
R
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut, (Biozentrum), Universität Würzburg, Germany.
Maier
E
E
Gentschev
I
I
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D004553:Electric Conductivity; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007473:Ion Channels / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D018272:Porins
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D018272:Porins; C073126:tolC protein, E coli