Molecular Insight of the Ice Nucleation by Ice Binding Protein: A Comparative Study With Ice Nucleating Protein, Antifreeze Protein, and Non-Ice-Binding Protein

Ice-binding proteins (IBPs) are a special class of proteins that can specifically interact with ice. A subclass of IBPs that can inhibit ice growth by depressing the freezing point of ice in a noncolligative manner are called antifreeze proteins (AFPs). The other subclass of IBPs that can enhance the ice nucleation process are called ice nucleating proteins (INPs). In this study, using molecular dynamics simulations, we induced ice formation and further compared the nucleation behaviors of AFP, INPs, and non ice binding proteins (NonIBPs). The formation of the initial stable ice cluster is found to be closer to the T-X-T motif of INP than that of AFP and it is farthest from the NonIBP. The fraction of ice molecules in the largest cluster is also found to be highest for INP. More importantly, the configurations of ice clusters formed in different independent runs are found to be strikingly similar for INP which signifies the crucial role of its ice-binding surface in controlling the process. The configurations of the formed ice in different independent runs are very dissimilar for both AFP and NonIBP. Quantitative analysis revealed that INP exhibits a stronger preference for hexagonal ice formation over its cubic counterpart compared to AFP/NonIBP. Between the two model INP structures used in this study, the latest model structure predicted by AlphaFold2 showed superior ice nucleating abilities than the older INP model. Similar conclusions about the abilities of ice nucleation by these different classes of proteins are confirmed by free energy calculation. Finally, the ice nucleating ability of the water-organizing motif of an INP is demonstrated over its nonwater-organizing motif in a single structure. Our findings provide clear evidence of INP’s ability to promote ice nucleation over AFPs and other NonIBPs.