Protonation-Regulated Membrane-Insertion Dynamics of pH Low-Insertion Peptide: Metastable Molecular Conformations and Their Transitions

The pH-triggered structural transition and translocation of the pH low-insertion peptide (pHLIP) across cell membranes, facilitated by its distinct protonation property, render it a valuable model for investigating the membrane insertion mechanism of molecules. This capability also holds significant promise for advancements in cancer diagnosis and transmembrane transport. In this study, we investigated the dynamics of membrane insertion of wild-type pHLIP and its three variants using real-time tracking of single-peptide translocation kinetics. We identified three distinct metastable molecular conformations of pHLIPs within the bilayer, referred to as ″kinetic intermediate states″ at varying depths within the bilayer. These metastable conformations were observed during both the pH-triggered membrane insertion process and at intervening pH levels (between 7.4 and 5.0). Over time following a decrease in pH, these molecular conformations gradually transitioned with an increasing number of peptides shifting from a horizontally bound state to an inserted state, with a gradual deepening of their depth until equilibrium was reached around 10 min. Additionally, all individual peptides within the membrane experienced subsecond level kinetic fluctuations. Modifications such as P20G increased penetration depth without affecting the insertion process, whereas truncating residues D and E from the C-terminal accelerated membrane insertion speed but reduced penetration depth. Our findings elucidate how residue protonation-driven conformational changes influence peptide dynamics during membrane insertion, thereby providing insights for designing advanced drug delivery systems.