Comparison of lipid-dependent bilayer insertion of pHLIP and its P20G variant

The ability of the pH-Low Insertion Peptide (pHLIP) to insert into lipid membranes in a transbilayer conformation makes it an important tool for targeting acidic diseased tissues. pHLIP can also serve as a model template for thermodynamic studies of membrane insertion. We use intrinsic fluorescence and circular dichroism spectroscopy to examine the effect of replacing pHLIP's central proline on the pH-triggered lipid-dependent conformational switching of the peptide. We find that the P20G variant (pHLIP-P20G) has a higher helical propensity than the native pHLIP (pHLIP-WT), in both water:organic solvent mixtures and in the presence of lipid bilayers. Spectral shifts of tryptophan fluorescence reveal that with both pHLIP-WT and pHLIP-P20G, the deeply penetrating interfacial form (traditionally called State II) is populated only in pure phosphocholine bilayers. The presence of either anionic lipids or phosphatidylethanolamine leads to a much shallower penetration of the peptide (referred to here as State IIS, for “shallow”). This novel state can be differentiated from soluble state by a reduction in accessibility of tryptophans to acrylamide and by FRET to vesicles doped with Dansyl-PE, but not by a spectral shift in fluorescence emission. FRET experiments indicate free energies for interfacial partitioning range from 6.2 to 6.8kcal/mol and are marginally more favorable for pHLIP-P20G. The effective pKa for the insertion of both peptides depends on the lipid composition, but is always higher for pHLIP-P20G than for pHLIP-WT by approximately one pH unit, which corresponds to a difference of 1.3kcal/mol in free energy of protonation favoring insertion of pHLIP-P20G. [Display omitted] •Membrane insertion of pHLIP is modulated by lipid composition.•The unstructured State II is only present in pure phosphatidylcholine membranes.•A new shallow and unstructured interfacial State IIS is identified.•Interfacial partitioning free energy for both peptides ranges from 6 to 7kcal/mol.•Protonation-dependent insertion ΔG is 1.3kcal/mol more favorable for pHLIP-P20G than pHLIP-WT.