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 (<i>T</i>_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.