Visualizing Ice-Binding Specificity of Designed Peptides via Fluorescence-Based Ice Plane Affinity (FIPA) for Cryopreservation Applications

Abstract

Cryopreservation requires inhibition of ice growth and recrystallization, where antifreeze proteins (AFPs) and peptides play crucial roles. To investigate how these molecules interact with ice, we employed Fluorescence-based Ice Plane Affinity (FIPA), enabling real-time visualization of peptide binding on crystallographic ice planes. We designed Fmoc-WW-based amphiphilic peptides with varying stereochemistry to modulate ice-binding specificity. FIPA analysis using single crystal ice aligned along a- and c-axes revealed that peptide chirality affects preferential binding to basal or prism planes. Peptide self-assembly morphology was characterized via TEM and Cryo-TEM to support structural understanding. These results suggest that chirality and sequence-controlled peptides can directionally regulate ice growth, providing a strategy to develop efficient, low-toxicity cryoprotectants that mimic or outperform natural AFPs.