Head-to-Tail Cyclization Enhances Ice-Growth Inhibition by Linear Threonine Oligomers

Developing short antifreeze peptides with low immunogenicity is considered to be a promising strategy for improving cryopreservation. Inspired by the design principles of cyclic peptide drugs characterized by high stability and strong affinity, we propose to use the cyclization strategy as a principle for the design of antifreeze peptides, aiming to enhance their structural stability and ice-binding ability, thereby significantly improving their antifreeze activity. In this study, we choose linear threonine oligomers (L-(Thr)), composed of common and biocompatible threonine residues, to investigate the mechanism and efficacy of cyclization. Molecular dynamics (MD) simulations are used to compare the ice-growth inhibition ability of a series of linear oligomers and their corresponding cyclic counterparts (49 molecular systems) on different ice planes, resulting in 80.8 μs MD trajectories. A detailed analysis of conformational changes during inhibition and their correlation with inhibitory efficiency reveals that conformational variability is detrimental to the binding of L-(Thr) to ice, while β-sheet-like conformation has a significant advantage in inhibiting ice growth and is identified as a key factor for the superior performance of cyclized oligomers (C-(Thr)) over their linear counterparts. Encouragingly, we find that C-(Thr)₁₂ exhibits the most prominent performance, surpassing previously reported cyclic peptides of similar size due to its enhanced structural stability, superior ice binding, coverage, and antiengulfment capabilities. This study provides valuable insights into the design of small-sized ice-growth inhibitors through head-to-tail cyclization of linear oligomers. However, it should be noted that our findings are based purely on computational simulations, and experimental validation in actual cryopreservation conditions remains necessary.