Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer

Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P-(alpha), a primary amphipathic cell-penetrating peptide which remains alpha-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P-(alpha) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying. at at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P-(alpha) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long. laments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P-(alpha). The simultaneous two-states organization of P-(alpha), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.