Alternative mechanisms for the interaction of the cell-penetrating peptides penetratin and the TAT peptide with lipid bilayers

Cell-penetrating Peptides (CPPs) have recently attracted much interest due to their apparent ability to penetrate cell membranes in an energy-independent manner. Here molecular-dynamics simulation techniques were used to study the interaction of two CPPs: penetratin and the TAT peptide with 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospolipid bilayers shed light on alternative mechanisms by which these Peptides might cross biological membranes. In contrast to previous simulation studies of charged Peptides interacting with lipid bilayers, no spontaneous formation of transmembrane pores was observed. Instead, the simulations suggest that the Peptides may enter the cell by micropinocytosis, whereby the Peptides induce curvature in the membrane, ultimately leading to the formation of small vesicles within the cell that encapsulate the Peptides. Specifically, multiple Peptides were observed to induce large deformations in the lipid bilayer that persisted throughout the timescale of the simulations (hundreds of nanoseconds). Pore formation could be induced in simulations in which an external potential was used to pull a single penetratin or TAT peptide into the membrane. With the use of umbrella-sampling techniques, the free energy of inserting a single penetratin peptide into a DPPC bilayer was estimated to be approximately 75 kJmol(-1), which suggests that the spontaneous penetration of single Peptides would require a timescale of at least seconds to minutes. This work also illustrates the extent to which the results of such simulations can depend on the initial conditions, the extent of equilibration, the size of the system, and the conditions under which the simulations are performed. The implications of this with respect to the current systems and to simulations of membrane-peptide interactions in general are discussed.