Proline-Mediated Conformational Rigidity Governs the Ice-Binding Behavior of Antifreeze Glycopeptides

Abstract

Antifreeze glycoproteins (AFGPs) are among the most potent inhibitors of ice recrystallization, yet the conformational basis of their activity and the effects of Proline mutations in short AFGP peptides remain unclear. Here, molecular dynamics simulations were used to examine the interfacial conformations and ice-growth inhibition of the minimal antifreeze peptide AFGP8 and its variants P7A, P13A, and P7A/P13A on three ice planes. Native AFGP8 exhibits a highly stable backbone and well-segregated hydrophobic/hydrophilic regions that are essential for its activity. The P7A mutation markedly increases flexibility and retains activity on the rough secondary prism face, while becoming fully inactive and rapidly engulfed on the primary prism face. In contrast, P13A and the double mutant P7A/P13A are more prone to being lifted or tilted by the ice surface, reducing surface coverage and interfacial stability. Overall, Pro7 and Pro13 cooperate to preserve the conformational rigidity and glycan geometry required for stable ice binding. These findings clarify the structural determinants underlying AFGP8 function, including the critical roles of its proline residues, and provide guidance for designing efficient biomimetic antifreeze agents.