Membrane permeation by NAF144-67 is determined by C-terminal electrostatics and N-terminal insertion

Cell penetrating peptides are short peptides that permeate cell membranes without the assistance of protein channels or pumps. NAF144-67 is an anti-cancer peptide that selectively permeates cancer cell membranes and permeates membranes to a greater extent when more negatively charged phospholipids are present in the phospholipid bilayer. Computational studies have shown that NAF144-67 directly traverses the membrane, assisted by membrane structural defects caused by the phospholipid headgroups. A mechanistic question is the order of events during permeation. Does the N-terminus permeate before the C-terminus, as suggested by the simulations? We attached the fluorophore dansyl to the N- and C-termini of NAF144-67. Fluorescence spectroscopy was used to probe whether each terminus of NAF144-67 interacts with the polar headgroup or hydrophobic tails of phospholipids in large unilamellar vesicles composed of phosphocholine, phosphoethanolamine, phosphoserine, sphingomyelin, and cholesterol with lipid compositions mimicking normal and cancer cell plasma membranes. To further examine mechanistic events during peptide translocation across membranes, three tryptophan mutants were prepared, and the depth of peptide insertion was evaluated by monitoring the extent of tryptophan fluorescence quenching using phosphocholine lipids brominated at various positions to probe the role of the glycerol backbone or phospholipid tails. We also quantified peptide permeation into normal and cancer cell membranes through a mass spectrometry-based trypsin cleavage assay. Molecular dynamics simulations predicted the location of specific residues within NAF144-67 when embedded in normal and cancer membranes. These methods demonstrate that NAF144-67 binds negatively charged phospholipid headgroups with its positively charged C-terminus, inserts its hydrophobic N-terminus into the lipid interior, and is selective toward cancer model membranes.