Mechanism of Action of pH-Triggered, Membrane Active Peptides

Abstract

The plasma membrane insulates a cell from its outside environment, serving as a selectively impermeable barrier across which only small or hydrophobic molecules can pass. Although the membrane is necessary for life, it is also problematic when useful macromolecules such as antibodies, peptides, polysaccharides, and imaging agents are blocked from entry. Most macromolecules can easily be uptaken by the cell through endocytosis, but remain trapped and eventually degraded within endosomes, which mature into lysosomes. To promote the escape of macromolecules from endosomes prior to their maturation into lysosomes, we used a high throughput screen to discover pH triggered, pore-forming peptides[1]. To determine their mechanism of action, we measured the peptides’ membrane binding affinity, secondary structure, and induction of pore-formation in POPC membranes. We identified that at least 5 acidic residues are essential for mediating a change from a soluble, predominantly unfolded, and inactive state at pH 7 to a membrane bound, helical, and active state at pH 5. These peptides are highly potent, with significant macromolecular leakage occurring at concentrations as low as 2 peptides per 1000 lipids. We determined that the peptides behave dynamically, associating with and dissociating from membranes, and consequently form pores on multiple vesicles. By atomic force microscopy imaging, we confirmed that the peptides form macromolecular pores, with diameters as large as 50 nm. Furthermore, we developed a kinetic model to explain how a low net fraction of bound peptide can lead to significant leakage. This work yields biophysical insights that will improve the design of peptides for new biomedical applications.[1]Wiedman, G., Kim, S.Y., Zapata-Mercado, E., Wimley, W.C. and Hristova, K., 2017. J. Am. Chem. Soc., 139(2), pp.937-945.