Figure 5.

Synthetic molecular evolution of potent equilibrium pore formers from melittin. A. The amphipathic helix of melittin was used to rationally vary specific residues in the melittin sequence. Residues maked with green were varied. B. The members of this library were screened using an assay that tests for equilibrium pore formation, rather than the transient pore formation that melittin causes. In this assay leakage of entrapped probes is measured as usual in Step 1. Later, access of a polar quencher to the vesicle interior at equilibrium is measured in Step 2. Only equilibrium pore formers enable access and ≥90% quenching. C. 10,000 library members were screened using this assay at a low peptide concentration. Most were inactive, as was melittin. D. A few potent equilibrium pore-formers were identified. Sequences of the best -studied examples are shown.