Despite ongoing research on antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs), their precise translocation mechanism remains elusive. This includes Buforin 2 (BF2), a well-known AMP, for which spontaneous translocation across the membrane has been proposed but a high barrier has been calculated. Here, we used computer simulations to investigate the effect of a nonequilibrium situation where the peptides are adsorbed on one side of the lipid bilayer, mimicking experimental conditions. We demonstrated that the asymmetric membrane adsorption of BF2 enhances its translocation across the lipid bilayer by lowering the energy barrier by tens of kJ mol-1. We showed that asymmetric membrane adsorption also reduced the free energy barrier of lipid flip-flop but remained unlikely even at BF2 surface saturation. These results provide insight into the driving forces behind membrane translocation of cell-penetrating peptides in nonequilibrium conditions, mimicking experiments.

• MeSH
• Adsorption MeSH
• Antimicrobial Peptides chemistry   pharmacology   MeSH
• Cell Membrane metabolism   chemistry   MeSH
• Antimicrobial Cationic Peptides chemistry   pharmacology   metabolism   MeSH
• Lipid Bilayers * chemistry   metabolism   MeSH
• Cell-Penetrating Peptides chemistry   metabolism   MeSH
• Proteins MeSH
• Molecular Dynamics Simulation MeSH
• Thermodynamics MeSH
• Terpenes chemistry   pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antimicrobial Peptides MeSH
• buforin II MeSH Browser
• Antimicrobial Cationic Peptides MeSH
• Lipid Bilayers * MeSH
• Cell-Penetrating Peptides MeSH
• Proteins MeSH
• Terpenes MeSH

Cell membranes act as semi-permeable barriers, often restricting the entry of large or hydrophilic molecules. Nonetheless, certain amphiphilic molecules, such as antimicrobial and cell-penetrating peptides, can cross these barriers. In this study, we demonstrate that specific properties of transmembrane proteins/peptides can enhance membrane permeation of amphiphilic peptides. Using coarse-grained molecular dynamics with free-energy calculations, we identify key translocation-enhancing attributes of transmembrane proteins/peptides: a continuous hydrophilic patch, charged residues preferably in the membrane center, and aromatic hydrophobic residues. By employing both coarse-grained and atomistic simulations, complemented by experimental validation, we show that these properties not only enhance peptide translocation but also speed up lipid flip-flop. The enhanced flip-flop reinforces the idea that proteins such as scramblases and insertases not only share structural features but also operate through identical biophysical mechanisms enhancing the insertion and translocation of amphiphilic molecules. Our insights offer guidelines for the designing of translocation-enhancing proteins/peptides that could be used in medical and biotechnological applications.

• MeSH
• Cell Membrane metabolism   chemistry   MeSH
• Hydrophobic and Hydrophilic Interactions * MeSH
• Lipid Bilayers chemistry   metabolism   MeSH
• Membrane Proteins * chemistry   metabolism   MeSH
• Molecular Dynamics Simulation * MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipid Bilayers MeSH
• Membrane Proteins * MeSH