Methionine Oxidation Stabilizes α-Helices but Attenuates Biological Activity in Amphipathic Peptides

The relationship between α-helicity and membrane activity is a central yet unresolved issue in the design of membrane-active peptides. While the amphipathic α-helical structure is widely regarded as a key determinant of antimicrobial activity, increasing evidence suggests that enhanced helicity does not necessarily translate into effective membrane disruption. Here, we investigate this structure-function relationship using a well-defined synthetic amphipathic α-helical scaffold, BKBA-20, to which methionine residues were introduced at specific locations and compared in their reduced and oxidized (methionine sulfoxide) forms. Methionine oxidation provides a unique means to modulate side-chain polarity without altering the peptide backbone, enabling experimental decoupling of α-helical stability from hydrophobic driving forces. Circular dichroism analyses revealed that oxidation to methionine sulfoxide increased α-helical content in aqueous buffer. Strikingly, however, oxidized peptides exhibited markedly attenuated antimicrobial and hemolytic activities, regardless of their sequence. In addition, the biological activities of the reduced peptides depended strongly on methionine placement, resulting in a clear positional bifurcation between antimicrobial-selective and variants exhibiting relatively higher, but still modest, hemolytic activity. These findings demonstrate that α-helicity is necessary but not sufficient for biological activity and that productive biological function is governed by overall hydrophobicity rather than secondary structure alone. The observed decoupling of helicity from biological activity is consistent with conceptual frameworks such as the interfacial activity model and highlights methionine oxidation as a molecular switch for tuning structure-function relationships. This work provides mechanistic insight into functional selectivity and offers design principles for redox-responsive biologically active peptides.