Translocation of Cationic Amphipathic Peptides Across Phospholipid Bilayers

Abstract

We have developed a method to measure translocation of amphipathic peptides across lipid membranes that uses giant unilamellar vesicles (GUVs) containing inner vesicles. When the GUVs are added to a peptide solution containing a water-soluble fluorescent dye, permeabilization of the membrane of the outer vesicle by the peptide results in the appearance of fluorescence in its lumen. The inner vesicles remain dark if the peptide does not translocate. However, the appearance of dye in the inner vesicles indicates peptide translocation across the outer membrane of the GUVs. This is because to cause dye flux into the inner vesicles, the peptide must have crossed the membrane of the outer vesicle. The method does not require that the peptide itself be fluorescently labeled; but if it is, this approach can be used to measure the kinetics of translocation. Initially we tested the method with three peptides derived from each class of antimicrobial, cytolytic, and cell-penetrating peptides. Those three peptides also had very different Gibbs energies of insertion into the bilayer, approximated by the difference between the Gibbs energy of binding to the membrane and the Gibbs energy of transfer from water to octanol. The initial measurements appeared to indicate that the probability of translocation was inversely correlated with the Gibbs energy of insertion. Now we extended this study to several variants of the original peptides. Two hypotheses were tested: (1) The probability of translocation simply decreases as the Gibbs energy of peptide insertion increases; (2) The probability of translocation increases if the distribution of positively charged residues along the peptide sequence is such that the inserted peptide has similar probabilities of returning the outside and crossing the membrane, to the inside of the vesicle. The results of these new tests are discussed.