Cooperative Ice Binding of Engineered Antifreeze Protein Produces Superior Activity

Antifreeze proteins (AFPs) bind irreversibly to ice surfaces and prevent ice crystal growth at temperatures of up to 10 οC below the melting point. The remarkable ability of AFPs to adsorb onto the ice surface and stop further growth is not fully understood, and their structural diversity hinders the effort to find a universal ice-binding motif. Multivalent AFP assemblies have achieved better ice growth inhibition compared to the corresponding monomers, yet the mechanism of these improved inhibitors is poorly understood. The innovative approach of this study was to test the effect of multivalency on the AFP’s adsorption rates to ice in addition to testing AFP activity. A monomer, dimer and multimer (12-subunits) of type III AFP were tested for thermal hysteresis (TH) activity and their adsorption rates were measured using fluorescence microscopy. To fit the experimental data, a revised Langmuir adsorption model was developed. As expected, the monomer achieved the lowest TH activity and its adsorption rate was slowest, followed by the dimer, which achieved slightly higher TH activity. The multimer was the most active and its adsorption rate was found to be 11-fold higher than that of the monomer. The newly developed model found cooperativity effects of the multimer, but not of the monomer and dimer. The rate at which the ice surface is covered by multimeric AFPs increases with continuous binding, suggesting cooperative ice binding. These results suggest a mechanism for AFP multimer binding. After binding of its first subunit to ice, subsequent binding of a second subunit becomes faster, which in turn facilitates the binding of a third subunit. Thus, cooperative ice binding is key for superior inhibition of ice growth.