Zbl. Bakt. 278, 187-196 (1993) © Gustav Fischer Verlag, StuttgartlNew York TolC of Escherichia coli Functions as an Outer Me111brane Channel ROLAND BENZ l , ELKE MAIER l, and IVA YLO GENTSCHEV 2 1 Lehrstuhl fUr Biotechnologie and 2 Lehrstuhl fiir Mikrobiologie, Theodor-Boveri-Institut (Biozentrum) der Universitat Wiirzburg, 8700 Wiirzburg, Germany With 4 Figures Summary Reconstitution experiments were performed with TolC from Escherichia coli outer mem- brane 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 experi- ments suggested that TolC formed ion-permeable channels. The channels of 80 pS in 1 M KCI 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 de- pendent on the bulk aqueous KCI 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. Zusammenfassung TolC von Escherichia coli wurde durch praparative SDS-Gelelektrophorese gereinigt. Oligomere, aber nicht Monomere bilden in kiinstlichen Lipidmembranen kleine ionen- permeable Kanale, die mit 80 pS in 1 M KCI eine wesentlich kleinere Einzelkanalleitfahig- keit haben als die generellen Diffusionsporen der auBeren Membran (1500 pS). Die Einzel- kanalleitfahigkeit war nur maBig von def KCI-Konzentration in der wassrigen Phase abhan- gig. Die durch TolC induzierte Membranleitfahigkeit konnte durch die Zugabe von Pepti- den zur wassrigen Phase reduziert werden, was die Anwesenheit einer Bindestelle fiir Peptide in dem Kanal andeutet. Die Dissoziationskonstante fiir die Bindung des Tripeptids H-Gly- Gly-Leu-OH an die Bindestelle ist etwa 20 mM. Die Ergebnisse weisen darauf hin, daB TolC eine AuBenmembranpore fiir Peptide ist.