Rational Modulation of pH-triggered Macromolecular Poration by Peptide Acylation and Dimerization.

The synthetically evolved pH-dependent delivery (pHD) peptides are a unique family of peptides that bind to membranes, fold into α-helices, and form macromolecule-sized pores at low concentration in response to acidic pH < 6. They have potential applications in drug delivery and tumor targeting but their activity and membrane selectivity must be optimized. Here, we develop a better understanding of how pHD peptide activity in membranes can be modulated by increasing hydrophobicity and membrane binding without changing amino acid sequence. We increased hydrophobicity of a representative pHD peptide, pHD108 (GIGEVLHELAEGLPELQEWIHAAQQLGC-amide) by coupling acyl groups of 6-16 carbons to the N- or C-termini and also by forming peptide dimers linked through a C-terminal cysteine residue. Unlike unmodified peptide, almost all variants formed oligomers in buffer and folded into α-helices both at pH 5 and at pH 7. Despite this, all variants partitioned strongly into fluid phase PC vesicles at pH 5 and at pH 7. Most variants enabled potent macromolecular poration, showing that the unique structure of the large pHD peptide pore can form when peptide termini are tethered to the bilayer with acyl chains, or when the peptide is dimerized at the C-terminus. Activity of acylated variants did not retain pH sensitivity; they were highly active at both pH 7 and pH 5 because they are membrane bound at both pH values, while the unmodified peptide is not active at pH 7 because it is not bound. The pHD peptide dimer retained some pH sensitivity. At pH 5 the dimer is the most active peptide studied in this work, with macromolecular poration occurring at a peptide to lipid ratio of 1:2000. These results confirm that membrane binding - rather than pH - is the predominant factor in activity, while also showing that acylation on either termini or dimerization are viable methods to modulate pHD108 activity. Finally, we add experimental constraints to possible pore architecture by showing little, if any, dependence of macromolecular poration on either pH or salt concentration, suggesting a lack of strong lateral electrostatic interactions between peptides in the pore. We therefore propose a toroidal pore structure, with peptides spanning the membrane in a parallel or mixed parallel and antiparallel orientation.