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.

Computational Design of Pore-Forming Peptides with Potent Antimicrobial and Anticancer Activities.

Peptides that form transmembrane barrel-stave pores are potential alternative therapeutics for bacterial infections and cancer. However, their optimization for clinical translation is hampered by a lack of sequence-function understanding. Recently, we have de novo designed the first synthetic barrel-stave pore-forming antimicrobial peptide with an identified function of all residues. Here, we systematically mutate the peptide to improve pore-forming ability in anticipation of enhanced activity. Using computer simulations, supported by liposome leakage and atomic force microscopy experiments, we find that pore-forming ability, while critical, is not the limiting factor for improving activity in the submicromolar range. Affinity for bacterial and cancer cell membranes needs to be optimized simultaneously. Optimized peptides more effectively killed antibiotic-resistant ESKAPEE bacteria at submicromolar concentrations, showing low cytotoxicity to human cells and skin model. Peptides showed systemic anti-infective activity in a preclinical mouse model of Acinetobacter baumannii infection. We also demonstrate peptide optimization for pH-dependent antimicrobial and anticancer activity.

Binding of DEP domain to phospholipid membranes: More than just electrostatics.

Over the past decades an extensive effort has been made to provide a more comprehensive understanding of Wnt signaling, yet many regulatory and structural aspects remain elusive. Among these, the ability of Dishevelled (DVL) protein to relocalize at the plasma membrane is a crucial step in the activation of all Wnt pathways. The membrane binding of DVL was suggested to be mediated by the preferential interaction of its C-terminal DEP domain with phosphatidic acid (PA). However, due to the scarcity and fast turnover of PA, we investigated the role on the membrane association of other more abundant phospholipids. The combined results from computational simulations and experimental measurements with various model phospholipid membranes, demonstrate that the membrane binding of DEP/DVL constructs is governed by the concerted action of generic electrostatics and finely-tuned intermolecular interactions with individual lipid species. In particular, while we confirmed the strong preference for PA lipid, we also observed a weak but non-negligible affinity for phosphatidylserine, the most abundant anionic phospholipid in the plasma membrane, and phosphatidylinositol 4,5-bisphosphate. The obtained molecular insight into DEP-membrane interaction helps to elucidate the relation between changes in the local membrane composition and the spatiotemporal localization of DVL and, possibly, other DEP-containing proteins.