Optimizing properties of translocation-enhancing transmembrane proteins.

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.

Insertases scramble lipids: Molecular simulations of MTCH2.

Scramblases play a pivotal role in facilitating bidirectional lipid transport across cell membranes, thereby influencing lipid metabolism, membrane homeostasis, and cellular signaling. MTCH2, a mitochondrial outer membrane protein insertase, has a membrane-spanning hydrophilic groove resembling those that form the lipid transit pathway in known scramblases. Employing both coarse-grained and atomistic molecular dynamics simulations, we show that MTCH2 significantly reduces the free energy barrier for lipid movement along the groove and therefore can indeed function as a scramblase. Notably, the scrambling rate of MTCH2 in silico is similar to that of voltage-dependent anion channel (VDAC), a recently discovered scramblase of the outer mitochondrial membrane, suggesting a potential complementary physiological role for these mitochondrial proteins. Finally, our findings suggest that other insertases which possess a hydrophilic path across the membrane like MTCH2, can also function as scramblases.

Peptide translocation across asymmetric phospholipid membranes.

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.

Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase.

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identify the Voltage Dependent Anion Channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act to import lipids into mitochondria.

Insertases Scramble Lipids: Molecular Simulations of MTCH2.

Scramblases play a pivotal role in facilitating bidirectional lipid transport across cell membranes, thereby influencing lipid metabolism, membrane homeostasis, and cellular signaling. MTCH2, a mitochondrial outer membrane protein insertase, has a membrane-spanning hydrophilic groove resembling those that form the lipid transit pathway in known scramblases. Employing both coarse-grained and atomistic molecular dynamics simulations, we show that MTCH2 significantly reduces the free energy barrier for lipid movement along the groove and therefore can indeed function as a scramblase. Notably, the scrambling rate of MTCH2 in silico is similar to that of VDAC, a recently discovered scramblase of the outer mitochondrial membrane, suggesting a potential complementary physiological role for these mitochondrial proteins. Finally, our findings suggest that other insertases which possess a hydrophilic path across the membrane like MTCH2, can also function as scramblases.

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins.

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.