Bottom-up design of efficient antifreeze peptides

Abstract

The rational design and development of antifreeze peptides remain significantly challenging. This study introduces an innovative strategy for the bottom-up design of efficient antifreeze peptides, coupled with a comprehensive mechanistic analysis. The critical amino acid glycine (G) was identified in AFPs from snow fleas, spiders, and silkworms. Efficient screening of the dipeptide ice-binding motif (GX) revealed glycine–glutamic acid (GE) as the optimal unit, which exhibits exact lattice matching (7.45 Å) with an ice crystal. The GE motif can endow polyethylene glycol (PEG)—a typical non-antifreeze molecule—with antifreeze activity. Furthermore, an efficient peptide—GEGGGGSNNT (GE-NNT)—was engineered by integrating a highly hydrophilic stabilization motif (NNT) via a flexible linker. GE-NNT demonstrated improvements of 45.7% and 61.4% over GE in inhibiting ice-crystal growth (9.0 µm s⁻¹) and ice recrystallization (21%), respectively, at a concentration of 1 mg mL⁻¹. Molecular dynamics (MD) simulations revealed that the NNT motif facilitates the formation of an ice-like hydration network. This work identifies a minimalistic ice-binding motif, GE, and uses it to design an efficient antifreeze peptide from the bottom-up by integrating it with a hydrophilic motif, providing fundamental tools and insights for the development of antifreeze materials.