PORE FORMATION BY NISIN INVOLVES TRANSLOCATION OF ITS C-TERMINAL PARTACROSS THE MEMBRANE

Nisin is an amphiphilic peptide with a strong antimicrobial activity a gainst various Grampositive bacteria. Its activity results from permea bilization of bacterial membranes, causing efflux of cytoplasmic compo unds, To get information on the molecular mechanism of membrane permea bilization, a mutant of nisin Z containing the C-terminal extension As p-(His)(6) was produced. The biological and anionic lipid-dependent me mbrane activity of this peptide was very similar to that of nisin Z. A nalysis of the pH dependence of model membrane interactions with the e longated peptide indicated the importance of electrostatic interaction s of the C-terminus with the target membrane for membrane permeabiliza tion. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in whi ch trypsin was encapsulated in the lumen of large unilamellar vesicles . The results show that the C-terminal part of the peptide translocate s across model membranes, The pH and anionic lipid dependence of trans location closely paralleled the results of membrane permeabilization s tudies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capa city to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.