Modular Antifreeze Peptides: Sequence-Driven Control of Ice Recrystallization and Hydration Dynamics

The rational design of antifreeze peptides remains a significant challenge. In this study, we developed a series of short peptides with antifreeze activity: G-1 (GPACCCGPA), G-2 (GPACCCGPA)₂, and G-3 (GPACCCGPA)₃, by mimicking the repetitive sequences and structural motifs of natural antifreeze proteins (AFPs). Notably, antifreeze activity was enhanced with increasing peptide length and molecular weight, with G-3 exhibiting the most pronounced ice recrystallization inhibition. Systematic assays, including splat cooling, sucrose sandwich, and nanoliter osmometry, revealed that peptide elongation markedly suppressed ice growth rates without inducing thermal hysteresis (T_H). Structural analyses demonstrated that increased peptide length enhanced α-helical content and promoted the formation of ordered nanostructures with moderate aggregation, enabling multiple ice-binding motifs to interact directly with the ice-water interface. The combined results of water spectral analysis and molecular dynamics simulations further revealed that peptides modulated the organization of surrounding water clusters by increasing local hydrogen-bond ordering while disrupting long-range hydrogen-bond dynamics. This dual modulation weakened the integrity of the extended hydrogen-bond network in bulk water and delayed the transition into a highly ordered crystalline ice phase. Mechanistically, G-3 achieves dual functionality by coupling efficient initial ice templating with strong inhibition of crystal growth, providing a clear rationale for its superior activity. Furthermore, the peptides exhibited excellent biocompatibility and significantly improved post-thaw cell viability. Overall, the design strategy proposed in this study highlights the potential of modular peptide engineering for developing biocompatible, tunable cryoprotectants, offering mechanistic insights into peptide-water interactions and clarifying the molecular basis of peptide-mediated antifreeze activity.