Antifreeze proteins (AFPs) bind irreversibly to ice surfaces and prevent ice crystal growth at temperatures of up to 10 °C 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 efforts 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 is 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, we developed a revised Langmuir adsorption model. 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 identifies cooperativity effects in the multimer but not in 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 the AFP multimer binding. After the 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 to the superior inhibition of ice growth.
Shalom et al. (Fri,) studied this question.