Using Enhanced Sampling Molecular Dynamics Techniques to Probe the Thermodynamics of pHLIP Peptide Insertion into a Model Lipid Bilayer

Abstract

The molecular mechanism of spontaneous polypeptide folding and insertion into a membrane, as well as its exit and unfolding, is of interest from several perspectives, including the action of antimicrobial peptides, the folding and degradation of membrane proteins, and medical applications of pH-triggered insertion peptides. A notable example is pH (Low) Insertion Peptide (pHLIP), which is a water-soluble polypeptide derived from helix C of bacteriorhodopsin that has the ability to insert into a membrane at acidic pH to form a stable transmembrane α-helix. The insertion process takes place in three stages: pHLIP is unstructured and soluble in water at neutral pH(state I), unstructured and bound to the surface of a membrane at neutral pH(state II), and inserted into the membrane as an α-helix at low pH(state III). Our hypothesis is that, there is a connection between the free energy change of binding /insertion and pKa of insertion. To test this hypothesis, we used enhanced sampling molecular dynamics (MD) techniques (steered MD and umbrella sampling) to investigate the molecular interactions of a C-terminal-truncated pHLIP variant, pHLIP-1, with a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The goal is to characterize the free energy of binding from state I to state II and the free energy of insertion from state II to state III (α-helical peptide insertion) and to identify key structural aspects that govern these processes. Our results agree well with the previous experiments, providing the first steps in establishing a direct relationship between the thermodynamics and pKa of pHLIP peptide insertion.