Membrane composition reshapes the folding landscape of a pH-responsive peptide

Lipid composition drives membrane protein sorting, interactions, and function, but the precise mechanistic influence of the membrane on protein free energy landscapes remains largely unresolved. In this study, we probe how lipids reshape the folding landscape of the pH-low insertion peptide (pHLIP) using a combination of surface-enhanced and ultrafast two-dimensional infrared (2D IR) spectroscopies. The membrane composition has a direct effect on the peptide’s structural transitions: anionic phosphatidylserine lipids promote a more efficient, rigid insertion, triggering α-helical folding at higher pH and bypassing partially-folded intermediates. In contrast, neutral membranes enforce a pathway with more distinct intermediates, marked by prolonged surface-bound states. We also demonstrate that this process is bidirectional, where the peptide insertion actively remodels the membrane, disrupts lipid packing and enhances water penetration. Together, these results indicate that the lipid bilayer functions as a dynamic, responsive energy landscape that not only guides folding but also adapts to it. This framework advances our understanding of how biological membranes modulate co-translational folding and mitigate misfolding in vivo.