Energy Barriers and Helix Plasticity in the Membrane Insertion of pHLIP

Abstract

The pH (low) insertion peptide (pHLIP) is a 36-aa monomeric peptide which is both soluble in water and able to insert as a transmembrane helix in lipid membranes at low pH. Thus, pHLIP has three major states with characteristic secondary structure: it is unstructured in solution (state I) and when bound to the surface of lipid membranes at neutral pH (state II). However, it forms a transmembrane helix in membranes at acid pH (state III), with a pKa of insertion between states II and III of 6.0. The lipid insertion of pHLIP is mediated by the protonation of at least two Asp residues. Thus, pHLIP has to deal with the translocation of acidic residues through the membrane to insert.Here, we designed several mutant peptides where the number of aspartic residues in the hydrophobic region of pHLIP was modified. Some mutations altered peptide behavior in solution and their interaction with lipid. At the same time, we observed that there was an apparent linear relationship between the number of Asp and both the observed pKa and the cooperativity of the insertion and/or folding in the membrane.In order to study the role of transmembrane helix formation in the lipid insertion of pHLIP, we designed a mutant peptide where the key residue Pro20 had been mutated to Gly. This peptide retained the overall properties of pHLIP, however both the interfacial (II) and the transmembrane (III) states had a higher helical content than wt pHLIP, and the pKa of insertion was also higher.Our data suggest that i) the number of Asp and their location at the water-lipid interface affect the pKa and/or cooperativity of the transition and ii) the formation of the membrane interfacial helix promotes peptide insertion into the membrane.