Improved Protocol to Tackle the pH Effects on Membrane-Inserting Peptides.

Many important biological pathways rely on membrane-interacting peptides or proteins, which can alter the biophysical properties of the cell membrane by simply adsorbing to its surface to undergo a full insertion process. To study these phenomena with atomistic detail, model peptides have been used to refine the current computational methodologies. Improvements have been made with force-field parameters, enhanced sampling techniques to obtain faster sampling, and the addition of chemical-physical properties, such as pH, whose influence dramatically increases at the water/membrane interface. The pH (low) insertion peptide (pHLIP) is a peptide that inserts across a membrane bilayer depending on the pH due to the presence of a key residue (Asp14) whose acidity-induced protonation triggers the whole process. The complex nature of these peptide/membrane interactions resulted in sampling limitations of the protonation and configurational space albeit using state-of-the-art methods such as the constant-pH molecular dynamics. To address this issue and circumvent those limitations, new simulations were performed with our newly developed pH-replica exchange method using wild-type (wt)-pHLIP in different 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine membrane sizes. This technique provided enhanced sampling and allowed for the calculation of more complete Asp14 pKa profiles. The conformational heterogeneity derived from strong electrostatic interactions between Asp14 and the lipid phosphate groups was identified as the source of most pKa variability. In spite of these persistent and harder-to-equilibrate phosphate interactions, the pKa values at deeper regions (6.0-6.2) still predicted the experimental pK of insertion (6.0) since the electrostatic perturbation decays as the residue inserts further into the membrane. We also observed that reducing the system size leads to membrane deformations where it increasingly loses the ability to accommodate the pHLIP-induced perturbations. This indicates that large membrane patches, such as 256 or even 352 lipids, are needed to obtain stable and more realistic pHLIP/membrane systems. These results strengthen our method pKa predictive and analytical capabilities to study the intricate play of electrostatic effects of the peptide/membrane interface, granting confidence for future applications in similar systems.