title: Synthetic molecular evolution of pore-forming peptides by iterative combinatorial library screening.
abstract: We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have β-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not α-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic α-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.
authors: Department of Biochemistry and Molecular Biology SL43, Tulane University School of Medicine, New Orleans, LA 70112, USA.
authors: Aram J
authors: AJ
authors: Krauson
authors: Jing
authors: J
authors: He
authors: Andrew W
authors: AW
authors: Wimley
authors: Andrew R
authors: AR
authors: Hoffmann
authors: William C
authors: WC
authors: Wimley
mesh_terms: D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques; D019143:Evolution, Molecular; D057166:High-Throughput Screening Assays; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid
chemical_list: D010455:Peptides
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