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

The transport of molecules across cell membranes is vital for proper cell function and effective drug delivery. While most cell membranes naturally possess an asymmetric lipid composition, research on membrane transport predominantly uses symmetric lipid membranes. The permeation through the asymmetric membrane is then calculated as a sum of the inverse permeabilities of leaflets from symmetric bilayers. In this study, we examined how two types of amphiphilic molecules translocate across both asymmetric and symmetric membranes. Using computer simulations with both coarse-grained and atomistic force fields, we calculated the free energy profiles for the passage of model amphiphilic peptides and a lipid across various membranes. Our results consistently demonstrate that while the free energy profiles for asymmetric membranes with a small differential stress concur with symmetric ones in the region of lipid headgroups, the profiles differ around the center of the membrane. In this region, the free energy for the asymmetric membrane transitions between the profiles for two symmetric membranes. In addition, we show that peptide permeability through an asymmetric membrane cannot always be predicted from the permeabilities of the symmetric membranes. This indicates that using symmetric membranes falls short in providing an accurate depiction of peptide translocation across asymmetric membranes.

• MeSH
• Cell Membrane chemistry   MeSH
• Phospholipids * MeSH
• Lipid Bilayers * chemistry   MeSH
• Peptides MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phospholipids * MeSH
• Lipid Bilayers * MeSH
• Peptides MeSH

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.

• MeSH
• Cell Membrane MeSH
• Phospholipids MeSH
• Lipid Bilayers * MeSH
• Membrane Proteins MeSH
• Peptides * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phospholipids MeSH
• Lipid Bilayers * MeSH
• Membrane Proteins MeSH
• Peptides * MeSH

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.

• Keywords
• antibiotics, fluorescent probes, membrane structure, membrane transport, molecular biophysics, none, structural biology,
• MeSH
• Models, Biological MeSH
• Cell Membrane chemistry   metabolism   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Antimicrobial Cationic Peptides chemistry   metabolism   MeSH
• Protein Conformation * MeSH
• Monte Carlo Method MeSH
• Models, Molecular MeSH
• Porins chemistry   metabolism   MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antimicrobial Cationic Peptides MeSH
• Porins MeSH

We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle x-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies, both peptides, as well as their equimolar mixture, were found to remain upon adsorption in a surface-aligned topology and to induce significant membrane perturbation, as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so-called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with the formation of parallel heterodimers at concentrations below a synergistic increase of dye leakage from vesicles. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup region as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations.

• MeSH
• Cell Membrane metabolism   MeSH
• Dimerization MeSH
• Phosphatidylethanolamines MeSH
• Phosphatidylglycerols MeSH
• Antimicrobial Cationic Peptides metabolism   MeSH
• Lipid Bilayers chemistry   MeSH
• Lipids chemistry   MeSH
• Magainins metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-palmitoyl-2-oleoylphosphatidylethanolamine MeSH Browser
• 1,2-dioleoyl-sn-glycero-3-phosphoglycerol MeSH Browser
• Phosphatidylethanolamines MeSH
• Phosphatidylglycerols MeSH
• Antimicrobial Cationic Peptides MeSH
• Lipid Bilayers MeSH
• Lipids MeSH
• Magainins MeSH
• peptide-Gly-Leu-amide MeSH Browser