Membrane-Active Peptides: Stable Pore-Forming or Cell-Penetrating Peptides Selected With Orthogonal High-Throughput Screening

Abstract

There are numerous distinct mechanisms by which a peptide can interact with a lipid bilayer membrane and affect its structure or function. Interfacially-active peptides (i.e. antimicrobial or cell penetrating) partition into the interface and drive rearrangements in the lipids, such that the segregation between the hydrocarbon core and the interfacial zone is broken down. Stable pore-forming peptides assemble into long-lived transmembrane pores using the constraints imposed by the bilayer to direct self-assembly. (Only a few examples of true stable pore forming peptides are known.) We have developed two high throughput screens using lipid bilayer vesicles that simultaneously allow for detection of different membrane activities (i.e. orthogonal screening) and have successfully used them to screen combinatorial peptide libraries for very specific membrane activities. In our translocation screen, we simultaneously measure leakage from lipid vesicles, and the ability of a peptide to be cleaved by a vesicle-entrapped protease. Using this screen we identified 12 very potent membrane-penetrating peptides from a library of 13,000 members. These peptides, which share a common sequence motif, spontaneously and rapidly translocate across bilayers without inducing leakage of entrapped contents. These peptides also rapidly translocate across the plasma membranes of living cells without cell permeabilization or toxicity. In our stable pore screen we measure immediate leakage of vesicle contents upon addition of peptides, and then also for the continued existence of pores in the same vesicles after overnight incubation. The vast majority of so called “pore forming peptides” do not form stable pores in membranes; leakage is a transient phenomenon. However, using this screen we have identified stable pore formers among known peptides, including melittin. This orthogonal screen has also been used to identify true stable pore forming peptides in several peptide libraries.