Residue-specific structures and membrane locations of pH-low insertion peptide by solid-state nuclear magnetic resonance

The pH-low insertion peptide (pHLIP) binds to a membrane at pH 7.4 unstructured but folds across the bilayer as a transmembrane helix at pH∼6. Despite their promising applications as imaging probes and drug carriers that target cancer cells for cytoplasmic cargo delivery, the mechanism of pH modulation on pHLIP-membrane interactions has not been completely understood. Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy. Data on residue-specific conformation and membrane location describe pHLIP in various surface-bound and membrane-inserted states at pH 7.4, 6.4 and 5.3. The critical membrane-adsorbed state is more complex than previously envisioned. At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II′ that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.