The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that havevery similar sequences, hydrophobicities,
and amphipathicities but differ markedly in their net charge
and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic
and very potent againstmicroorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral proteinreconstruction
and peptide analogue design to examine the roles of electrostatic
and hydrophobic interactionsin the biological activity
and mode of action of functionally divergent Gly-Leu-rich peptides. The structure
and interaction of the peptides with anionic
and zwitterionic model membranes were investigated bycircular dichroism with 2-dimyristoyl-
sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-
sn-glycero-3-phosphatidylglycerol vesicles
and surface plasmon resonance with immobilized bilayers. The results,combined with antimicrobial assays, the kinetics of bacterial killing,
and membrane permeabilization assays,reveal that Gly, Val, Thr,
and Ile can all be accommodated in an amphipathic
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helix when the helix isin a membrane environment. Binding to anionic
and zwitterionic membranes fitted to a 2-stage interactionmodel (adsorption to the membrane followed by membrane insertion). The first step is governed byhydrophobic interactions between the nonpolar surface of the peptide helix
and the membranes. The strongbinding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step
andinvolves short-range Coulombic interactions that prolong the residence time of the membrane-insertedpeptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutionsto generate an orthologous cationic variant of neutral hemolytic peptides that bind to
and permeate bacterialcell membranes.