Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Abstract

The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric alpha-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I-state II transition) at 37 degrees C is approximately -7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II-state III transition) is nearly -2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.

Fig. 1.

Thermodynamic data on pHLIP interaction with POPC vesicles. (A–C) Scatchard plots constructed from the titration of pHLIP with POPC vesicles at pH 8.0 (A) and pH 5.0 (B) monitored by changes of tryptophan fluorescence and (C) by changes of heat release measured by ITC (titration data presented in Figs. S1 a, b, and f, respectively). (D) The pH-dependence of the heat effects induced by the pH changes of pHLIP-POPC. (E) The changes of enthalpy calculated from data in D over the course of the pHLIP transition from the denatured to the native state in lipid bilayers. (F) The pH-dependence of the logarithmic ratio of protonated to deprotonated forms of pHLIP. These plots were used to calculate the number of protons necessary to induce the transition. Red, 15°C; green, 22°C; blue, 37°C.

Fig. 2.

A schematic representation of pHLIP interaction with a lipid bilayer is shown. (A) In state I, the peptide is in solution at neutral and basic pHs. By addition of vesicles, the unstructured peptide is adsorbed on the membrane surface. State II, type I interactions occur at a low lipid/peptide ratio, when the total accessible membrane surface is not enough to accommodate the full length of peptide on it. We reason that the hydrophobic motif near the C terminus is adsorbed first (red sequence in B). By addition of more vesicles, the transition to the type II (state II) interaction is seen, at a high lipid/peptide ratio when there is enough accessible space on a membrane for peptides to freely occupy lipid surface area without competing with each other. In state III, a drop of pH leads to the protonation of Asp residues, increasing peptide hydrophobicity, and resulting in the insertion and formation of a transmembrane α-helix. Our thermodynamic measurements suggest the existence of three major populations of lipids: (i) lipids interacting with the peptide directly (lipids with blue head groups); (ii) lipids not interacting with the peptide directly but influenced by the interaction (lipids with cyan head groups, possibly in both leaflets); and (iii) lipids that are not involved in the interaction with pHLIP (lipids with yellow head groups). The values for energy changes are taken from Table 1 (for 37°C). (B) The pHLIP sequence is presented, with the presumed transmembrane part in bold (based on the helix seen in bacteriorhodopsin). Red is used to denote the hydrophobic sequence and the C terminus, which are expected to initially interact with the bilayer.