Multiscale mapping of antifreeze peptide binding at ice crystal interfaces across macro-to-atomic length scales

Abstract

Antifreeze proteins (AFPs) regulate ice growth by selectively adsorbing onto specific crystal planes of ice, effectively arresting recrystallization at the molecular level. Despite their remarkable interfacial activity, AFPs are structurally complex, costly to produce, and difficult to scale, limiting their practical utility as cryoprotectants. Short AFP-mimetic peptides offer a tractable alternative, yet a mechanistic understanding of how peptide molecular structure and supramolecular assembly govern ice crystal plane selectivity and interfacial behavior across multiple length scales remains lacking. Here, we report the design of short AFP-mimetic peptides that recapitulate ice plane-selective binding through defined molecular structures and controllable supramolecular assembly. To comprehensively elucidate how these peptides interact with ice crystal interfaces — from macroscopic crystal morphology down to atomicscale binding geometry — we employ a fully integrated multiscale characterization framework spanning macro, meso, nano, and atomic length scales. At the macroscopic scale, fluorescence-based ice plane affinity (FIPA) analysis maps the binding preferences of fluorescently labeled peptides across distinct ice crystal planes, revealing sequence-dependent selectivity that shifts systematically with peptide length. At the mesoscale, TEM and cryo-TEM resolve the self-assembled nanostructures formed by the peptides in aqueous and PBS buffer conditions, establishing a direct relationship between supramolecular organization and ice-binding behavior. At the nanoscale, interfacial structural characterization probes how peptide assemblies organize at the ice surface and mediate crystal plane recognition. At the atomic scale, molecular dynamics simulations and highresolution structural analysis resolve the specific intermolecular interactions and geometric complementarity that underpin plane-selective adsorption and ice growth inhibition. Collectively, this multiscale analysis demonstrates that ice crystal plane selectivity and interfacial activity are governed by a hierarchy of structural features — from atomic-level residue interactions to supramolecular assembly geometry — and that both can be rationally tuned through peptide sequence design. This work establishes a molecular design framework for AFP-mimetic peptides and provides mechanistic guidelines for the development of next-generation peptide-based cryoprotectants.