Bioinformatics‐Driven Design of Peptides for Membrane Stabilization During Cryopreservation

Abstract During cryopreservation, cooling triggers membrane phase transitions from a liquid‐crystalline to a gel phase, compromising membrane permeability, impairing water exchange, ultimately leading to cell death. Group 3 late embryogenesis abundant (G3LEA) proteins stabilize cell membranes under adverse circumstances through their functional 11‐mer repeats. Thus, we conducted a bioinformatics analysis of 11‐mer repeats across LEA proteins and identified AKE, a class A α‐helix peptide that lowers the gel‐to‐liquid crystalline phase transition temperature ( T m ). Structural optimization further established a key design principle: a class A α‐helix with a charge‐segregated structure, featuring two positively charged faces separated by hydrophobic and negatively charged regions, designed to enhance membrane interactions by promoting electrostatic binding to phospholipid head groups while allowing hydrophobic regions to associate with lipid tails, potentially strengthening overall membrane affinity. Results confirmed that substituting lysine with arginine, which carries a more delocalized and stable positive charge, strengthened electrostatic interactions and reduced free energy. The optimized peptide, ARE, lowered T m and reduced the extent of phase transition improved water permeability and osmotic resistance, leading to a 52% enhancement in post‐thaw red blood cell recovery. By integrating structural design with charge modulation, this study provides a framework for developing membrane stabilizers through rational peptide engineering.