Gain-of-function analogues of the pore-forming peptide melittin selected by orthogonal high-throughput screening

Abstract

We recently developed an orthogonal, high-throughput assay to identify peptides that self-assemble into potent, equilibrium pores in synthetic lipid bilayers. Here, we use this assay as a high-throughput screen to select highly potent pore-forming peptides from a 7776-member rational combinatorial peptide library based on the sequence of the natural pore-forming peptide toxin melittin. In the library we varied ten critical residues in the melittin sequence, chosen to test specific structural hypotheses about the mechanism of pore formation. Using the new high-throughput assay, we screened the library for gain-of-function sequences at a peptide to lipid ratio of 1:1000 where native melittin is not active. More than 99% of the library sequences were also inactive under these conditions. A small number of library members (0.1%) were highly active. From these we identified 14 potent, gain-of-function, pore-forming sequences. These sequences differed from melittin in only 2-6 amino acids out of 26. Some native residues were highly conserved and others were consistently changed. The two factors that were essential for gain-of-function were the preservation of melittin's proline-dependent break in the middle of the helix and the improvement and extension the amphipathic nature of the α-helix. In particular the highly cationic carboxyl-terminal sequence of melittin, is consistently changed in the gain-of-function variants to a sequence that it is capable of participating in an extended amphipathic α-helix. The most potent variants reside in a membrane-spanning orientation, in contrast to the parent melittin, which is predominantly surface bound. This structural information, taken together with the high-throughput tools developed for this work, enable the identification, refinement and optimization of pore-forming peptides for many potential applications.

Figure 1

The two step assay. top: Schematic diagram of the components of the two step assay. Unilamellar lipid vesicles are prepared with entrapped terbium and external dipicolinic acid (DPA) to probe leakage by measurement of complex formation (step one: leakage). In the same vesicles, access of the membrane impermeant quencher dithionite to the vesicle interior at equilibrium is measured by its ability to quench NBD dye-labeled lipids (step two: accessibility). Accessibility to the vesicle interior requires an equilibrium pore in the membrane. Bottom: Two step assay measurements of membrane permeabilizing peptides in 100% zwitterionic 1-palmitoyl-2-oleoyl,sn-gycero-3-phosphocholine (POPC) vesicles. Symbol sizes reflect total peptide concentration.

Figure 2

Top: The amino acid sequence of the pore-forming peptide melittin. Residues that were varied in the combinatorial library are shown. Bottom: A helical wheel projection of melittin showing the non-polar and polar faces of the helix. All residues are drawn in helical configuration. Orange symbols represent polar, uncharged residues. Blue symbols represent basic residues. Gray symbols represent hydrophobic residues. The extra one-letter residue codes shown on the helical wheel are the residues that were present in the combinatorial library. Each varied position also always including the native residue.

Figure 3

High-throughput screen results. A: Two step assay results for all 10,000 library members assayed. The screen was done at a nominal peptide:lipid ratio of 1:1000. Leakage and access of dithionite to NBD-lipids at equilibrium are plotted for each library member assayed. Under these conditions almost all library members are inactive with leakage near 0% and NBD quenching near 55%, indicating only surface exposed lipids are quenched. The area in the triangle at the upper right represents the most active peptides in the library with leakage and NBD-quenching at equilibrium near 100%. B: Histograms of the data plotted in panel A show that most library members are inactive under these conditions.

Figure 4

Sequences of 14 gain-of-function analogs identified in the high-throughput screen. The sequence of melittin and the variants present in the library are shown at the top. The screening results are shown at the bottom. The color code is green for conserved residues, blue for changed residues. Those that were excluded from the active variants are shown at the bottom.

Figure 5

Sequences of the peptides synthesized for further study. Residues shaded red are changed from the parent sequence of melittin. Residues in blue are the basic amino acids in the sequence. Mel-P1 and Mel-P2 are engineered single site variants to test the effect of the commonly observed T10A and K23A substitutions. Mel-P3 is the T10A, K23A double variant. Peptides Mel-P4 through Mel-P9 were actually observed in the screen. Mel-P4, 5 and 6 have T10A and K23A and different numbers of changed residues in the C-terminal tail. Mel-P7, 8 and 9 have other changes observed in the selection, such as V8G or lack the T10A substitution. “Δ” is the number of amino acid changes overall. “+” is the total charge of the peptide.

Figure 6

Verification of membrane permeabilization by selected and engineered peptides. Leakage of Tb³⁺/DPA was measured under conditions very similar to the high-throughput screen. In this experiment we used vesicles made from POPC and 10% POPG (as in the screen). Leakage from 1 mM vesicles was measured as a function of peptide concentration, and the peptide concentration that induces 50% leakage (LIC₅₀ or Leakage Inducing Concentration for 50%) was obtained by curve fitting. The engineered sequences differ from the parent by one or two commonly observed substitutions. The selected sequences were actually observed in the high-throughput screen.

Figure 7

Membrane permeabilization by selected and engineered peptides. Leakage of ANTS/DPX was measured by fluorescence. In this experiment we used vesicles made from various lipid compositions. Leakage from 1 mM vesicles was measured as a function of peptide concentration, and the peptide concentration that induces 50% leakage (LIC₅₀ or Leakage Inducing Concentration for 50%) was obtained by curve fitting.

Figure 8

Oriented circular dichroism. Top: Theoretical oriented CD spectra for 100% inserted (perpendicular) and 100% surface bound (parallel) α-helical peptides(53). Bottom: Melittin or one of the nine gain-of-function analogs were incorporated into stacked oriented multibilayers deposited on a quartz disc. Samples were enclosed in a chamber with a drop of water for hydration through the vapor phase and oriented CD spectra were taken. The lipid used was pure POPC and the P:L ratio was 1:200 for all samples.