Cell-penetrating peptides are efficient tools for intracellular delivery of a variety of cargoes. In this study, we explored the effect of chain length, side chain chemistry, and the locations of conjugated molecules on the interaction between iron-chelating peptides and a mitochondrial-mimicking membrane. We report that a longer chain length enhanced peptide/membrane interactions, and conjugation at the N-terminus lowered the free-energy barrier for peptide translocation across the membrane. Peptides containing Phe side chains and those containing modified Phe (cyclohexane) side chains showed comparable peptide/membrane energetics and translocation energy barriers. Using steered molecular dynamics (SMD) simulations, we further probed the mechanistic details of translocation of each N-terminated peptide across the membrane and compared their metastable states. At a higher steering velocity, the peptide adopted a compact structure due to frequent π-π interactions among conjugated molecules, but at lower steering velocities, each N-terminated peptide adopted an extended structure. This structure allowed cationic residues to maximize their interactions with phosphate headgroups in the mitomembrane. The hydrophobic residues also formed interactions with the lipid acyl tails, facilitating the passage of peptides across the membrane with decreased free energy barriers. Our results highlight the significance of peptide chain length and conjugation in facilitating peptide transport across the membrane.
