Janus amphiphilic organization as a shared structural basis for antifreeze and antimicrobial functions in short α-helical peptides.

Cryopreservation is challenged by both ice recrystallization damage and microbial contamination. Although antifreeze peptides (AFPTs) offer excellent sequence programmability and biocompatibility, integrating ice-growth inhibition and antimicrobial membrane disruption within a single short peptide scaffold remains a major molecular design challenge. Here, comparative structural analysis of collected α-helical AFPTs and α-helical antimicrobial peptides (AMPs) revealed a shared amphipathic α-helical organization characterized by directional radial segregation of hydrophilic and hydrophobic surfaces. We term this anisotropic architecture the Janus α-helix and propose it as a common interfacial framework for coupling antifreeze and antimicrobial functions. Guided by this framework, we reengineered a deep learning-identified AMP into P-Janus, a short α-helical peptide with enhanced Janus amphiphilicity. Molecular dynamics simulations and experiments demonstrated that P-Janus exhibits stable adsorption at the ice-water interface, suppresses ice recrystallization, and promotes persistent pore formation in bacterial membranes, thereby enhancing antimicrobial activity while maintaining low hemolytic activity. We further established a virtual screening workflow integrating Janus α-helix structural constraints with antifreeze activity prediction to mine natural AMP space. Combined computational and experimental validation identified five additional AMP-derived peptides with pronounced antifreeze activity. Comparison of these candidates revealed a graded structure-function relationship, in which stronger Janus-like radial face segregation generally promotes more effective interfacial recognition and antifreeze performance. Collectively, these findings establish the Janus α-helix as a transferable interfacial design principle for engineering short multifunctional peptides with integrated antifreeze and antimicrobial activities.