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PH-Triggered, Macromolecule-Sized Poration of Lipid Bilayers by Synthetically Evolved Peptides Gregory Wiedman, Sarah Y. Kim, Elmer Zapata-Mercado, William C Wimley, and Kalina Hristova J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b11447 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 21, 2016
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PH-Triggered, Macromolecule-Sized Poration of Lipid Bilayers by Synthetically Evolved Peptides Gregory Wiedman1,3, Sarah Y. Kim2.3, Elmer Zapata-Mercado2,3, William C. Wimley4*, and Kalina Hristova1,2,3* 1
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218 2
Graduate Program in Molecular Biophysics, Johns Hopkins University, Baltimore, MD 21218 3
Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218 4
Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, 70112.
*
Correspondence to
[email protected] and
[email protected] 1 ACS Paragon Plus Environment
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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 macromoleculesized 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.
INTRODUCTION Membrane-permeabilizing peptides could have utility in a variety of biotechnological and clinical applications due to their ability to breach the barrier imposed by lipid bilayers1-10. But to enable their practical application, they will first need to be designed to function only in specific environments, or only in response to specific triggers. One potentially useful trigger is pH, which varies in spatially and temporally specific ways in cellular organelles, and also varies locally in tissues under some pathological conditions, including cancer11-13. As an example application, pH-sensitive membrane permeabilizing peptides could be triggered upon endosomal acidification to promote the release of uptaken polar molecules from endosomal compartments into the cell cytosol1,14-16. Such an application would eliminate a long-standing barrier to the delivery of generic polar compounds, especially proteins and other macromolecules, to cells14,17,18. Indeed, while efficient methods exist to deliver oligonucleotides to cells16,19, most other types of macromolecules are more difficult to deliver. These macromolecules can be directed to existing 2 ACS Paragon Plus Environment
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cellular uptake mechanisms20,21, but in the absence of endosome permeabilization or disruption, they often get trapped within the classical pathways that lead to their lysosomal degradation or recycling without significant entry into the cytosol22,23. As a second example application, pHsensitive, membrane permeabilizing peptides could potentially be used in cancer therapies to selectively permeabilize the plasma membranes of cancer cells. This is possible because the environmental milieu in the vicinity of solid tumors is often acidic due to their high rate of mostly glycolytic metabolism13. In support of this application, the locally acidic pH of solid tumors has already been shown in mice to trigger the pH-sensitive insertion of peptides into membranes, although not for peptides that cause permeabilization11,24. Some progress has been made in the discovery or design of pH-triggered pore-forming peptides25,26, including some that we designed rationally27, and other pH triggered membrane active peptides16,28. The state-of-the art in the field has been trial and error-based addition of protonatable residues such as aspartate (D), glutamate (E) and histidine (H). Two well-studied examples are pHLIP, which inserts across membranes at pH
