The interfacial electrostatic potential modulates the insertion of cell-penetrating peptides into lipid bilayers

Cell-penetrating peptides (CPP) are short sequences of cationic amino-acids that show a surprising ability to traverse lipid bilayers. CPP are considered to be some of the most effective vectors to introduce membrane-impermeable cargos into cells, but the molecular basis of the membrane translocation mechanisms and its dependence on relevant membrane physicochemical properties have yet to be fully determined. In this paper we resort to Molecular Dynamics simulations and experiments to investigate how the electrostatic potential across the lipid/water interface affects the insertion of hydrophilic and amphipathic CPP into two-dimensional lipid structures. Simulations are used to quantify the effect of the transmembrane potential on the free-energy profile associated with the transfer of the CPP across a neutral lipid bilayer. It is found that the electrostatic bias has a relatively small effect on the binding of the peptides to the membrane surface, but that it significantly lowers the permeation barrier. A charge compensation mechanism, arising from the segregation of counter-ions while the peptide traverses the membrane, determines the shape and symmetry of the free-energy curves and underlines relevant mechanistic considerations. Langmuir monolayer experiments performed with a variety of amphiphiles model the incorporation of the CPP into the external membrane leaflet. It is shown that the dipole potential of the monolayer controls the extent of penetration of the CPP into the lipid aggregate, to a greater degree than its surface charge.