pH-Triggered, Macromolecule-Sized Poration of Lipid Bilayers by Synthetically Evolved Peptides

Abstract

pH-triggered membrane permeabilizing peptides could be exploited in a variety of applications, such as to enable cargo release from endosomes for cellular delivery, or as cancer therapeutics that selectively permeabilize the plasma membranes of malignant cells. Such peptides would be especially useful if they could enable the movement of macromolecules across membranes, a rare property in membrane permeabilizing peptides. Here we approach this goal by using an orthogonal high-throughput screen of an iterative peptide library to identify peptide sequences that have the following two properties: (i) little synthetic lipid membrane permeabilization at physiological pH 7 at high peptide concentration and (ii) efficient formation of macromolecule-sized defects in synthetic lipid membranes at acidic pH 5 and low peptide concentration. The peptides we selected are remarkably potent macromolecular sized pore-formers at pH 5, while having little or no activity at pH 7, as intended. The action of these peptides likely relies on tight coupling between membrane partitioning, α-helix formation, and electrostatic repulsions between acidic sidechains, which collectively drive a sharp pH-triggered transition between inactive and active configurations with apparent pKa values of 5.5–5.8. This work opens new doors to developing applications of peptides with membrane permeabilizing activities that are triggered by physiologically relevant decreases in pH.

Tuneable poration: host defense peptides as sequence probes for antimicrobial mechanisms

“…All such modifications, however subtle, confer resistance to HDPs. Conversely, antimicrobial sequences themselves present cryptic, but practical, strategies for circumventing evolving resistance mechanisms 64 . As it was long proposed 7 , developing a widespread resistance against HDPs, which are evolutionarily conserved molecules, would carry a high cost.…”

pH-Dependent Membrane Interactions of the Histidine-Rich Cell-Penetrating Peptide LAH4-L1

“…The transition is reversible and has been used to quantify the transfer energy of amino acid side chains from the membrane interface to the membrane interior (13,14). Since the development of LAH4 (1), an aspartate-rich peptide has been discovered that also exhibits pH-dependent membrane topology with potential biomedical applications (15), and a series of peptides designed to form pH-dependent macromolecular pores in membranes has been presented recently (16,17).…”

Unravelling cytosolic delivery of endosomal escape peptides with a quantitative endosomal escape assay (SLEEQ)

“…These peptides are thought to depend on acidification post-internalisation to induce endosomal escape and are commonly referred to as pH-dependent membrane-active peptides (PMAPs). pHlip is derived from bacteriorhodopsin 26,27 , whereas pHD118 is a derivitative of bee venom (melittin) 28 . The influenza haemagglutinin Nterminal peptide HA2 has also been investigated widely, with substitution of certain amino acids with glutamic acid providing improved pH dependent activity (such as E5) 29,30 .…”