Membrane Interactions Drive Hydrogen-Bond Dynamics in the pH-Low Insertion Peptide (pHLIP)

The pH Low Insertion Peptide (pHLIP), a cell-penetrating peptide, presents an ideal model to study peptide-membrane interactions across a range of conformational states. The folded, solvent-exposed unfolded, and membrane-inserted states of pHLIP have been well-characterized, but the intermediate structures remain poorly understood. Studies have focused on understanding folding and membrane interactions; however, there is a relation between the environment, membrane interactions, and local picosecond dynamics that has not been characterized. In this study, we use ultrafast two-dimensional infrared (2D IR) spectroscopy to investigate the combined secondary structure and local interfacial and membrane environments during the pH-induced insertion and folding processes of pHLIP into a phospholipid bilayer. Through a combination of amide I 2D IR spectroscopy, which measures structure and H-bond dynamics of the peptide backbone, and molecular dynamics (MD) simulations, we report on the intermediate states along the pH-driven insertion and folding structure and local environment dynamics of pHLIP into model POPC bilayers. The findings reveal that while a dynamic shift happens between pH 6.5 and 6.0, the α-helical transition occurs only between pH 6.0 and 5.8, well above the pH required for full insertion. The faster dynamics observed on this transition could be linked to disruptions in the hydrogen bond network during the partial folding. The results support the fold-then-insert mechanistic view of the pHLIP peptide.