Biology subjects

Aguilella-Arzo, M.

Publications and source records attributed to Aguilella-Arzo, M..

3 recordsLinked to original sources

bioRxiv2026

Membrane Lipid Composition and Asymmetry Act as a Switch for Cell-Penetrating Peptide Translocation versus Leakage

Cell-penetrating peptides (CPPs) can enable intracellular access while avoiding cytotoxicity, yet their behavior is highly sensitive to membrane composition. Here we show that lipid composition and leaflet asymmetry act as a switch that determines whether the amphipathic CPP MAP undergoes non-disruptive translocation or stabilizes membrane pores that drive leakage. Using computational electrophysiology (CompEL) simulations in membranes of increasing physiological relevance, we find that symmetric anionic bilayers favor MAP insertion coupled to transmembrane pore stabilization in POPC:POPG membranes, reproducing progressive dye release in liposome leakage assays. Incorporation of cholesterol reduces the number of inserted peptides yet enhances pore stabilization, consistent with faster leakage kinetics in cholesterol-containing vesicles. In contrast, an asymmetric membrane model mimicking the eukaryotic plasma membrane (POPC outer leaflet; POPC:POPS inner leaflet) supports MAP translocation without sustained membrane disruption. This delivery-relevant mechanism is supported by efficient MAP internalization in HEK293 cells while maintaining high viability. Polarized ATR-FTIR provides structural context, indicating predominantly membrane-associated -helical conformations across lipid compositions. Together, these results establish lipid asymmetry and composition as actionable biointerface parameters that tune CPP function between translocation and leakage and demonstrate an experimentally benchmarked framework for predicting membrane outcomes across complex lipid environments.

biophysics↗

bioRxiv2025

Predicting residue ionization of OmpF channel using Constant pH Molecular Dynamics as benchmarking

Electrostatic interactions play a key role in protein structure function. There is a large family of mesoscopic protein channels whose selectivity is mainly controlled by the protein electrostatic properties and ion specific channel interactions play a minor role. The knowledge of the charge state of the ionizable residues over a wide pH range, often summarized in their pKa, stands as the most valuable information for structure-function studies of many protein channels. However, experimental pKa determination is a difficult task, typically accomplished using Nuclear Magnetic Resonance only in a limited number of membrane proteins. Thus, the pKa calculation is the most frequently used alternative. Constant pH Molecular Dynamics (CpHMD) simulation provides arguably the most accurate pKa prediction method in proteins containing many charged residues since it captures the coupling between conformational dynamics and residue protonation. Here we study the charge state of a general diffusion porin, OmpF, in which protons exert a crucial regulation of the channel discrimination of small inorganic ions as well as antibiotic translocation. We examine the pKa prediction using different methods, with the CpHMD simulations as benchmarking, and discuss the somewhat unusual titration of several acidic residues. The most widely used pKa prediction methods, though useful for globular proteins, fail to capture the specificities of channel proteins embedded in biological membranes. This is the first attempt we know to use CpHMD to study the pH- dependent charge of a large multiionic channel (with over three hundred ionizable residues) embedded in a lipid membrane.

biophysics↗

bioRxiv2024

Computational Insights into Membrane Disruption by Cell-Penetrating Peptides

Cell-penetrating peptides (CPPs) can translocate into cells without inducing cytotoxicity. The internalization process implies several steps at different time scales ranging from microseconds to minutes. We combine adaptive Steered Molecular Dynamics (aSMD) with conventional Molecular Dynamics (cMD) to observe equilibrium and non-equilibrium states to study the early mechanisms of peptide- bilayer interaction leading to CPPs internalization. We define three membrane compositions representing bilayer sections, neutral lipids (i.e. upper leaflet), neutral lipids with cholesterol (i.e hydrophobic core), and neutral/negatively charged lipids with cholesterol (i.e. lower leaflet) to study the energy barriers and disruption mechanisms of Arg9, MAP, and TP2, representing cationic, amphiphilic, and hydrophobic CPPs, respectively. Cholesterol and negatively charged lipids increase the energetic barriers for peptide bilayer crossing. TP2 interacts with the bilayer by hydrophobic insertion, while Arg9 and MAP disrupt the bilayer by forming transient or stable pores. Collectively, these findings underscore the significance of innovative computational approaches in studying membrane-disruptive peptides, more specifically, in harnessing their potential for cell penetration.

biophysics↗