Growth suppression of ice crystal basal face in the presence of a moderate ice-binding protein does not confer hyperactivity

Significance Ice-binding proteins (IBPs) can be considered a prominent example of macromolecules affecting ice growth kinetics. Until now, two groups of IBPs have been described based on their activity as ice growth inhibitors: moderate and hyperactive IBPs. The mechanism underlying these activity differences has not been clarified yet. Although it is commonly believed that hyperactivity is related to growth inhibition of the basal faces of ice crystals, we show that a moderate IBP can also attach to the basal faces and inhibit their growth. Our observations clearly indicate that this moderate IBP occupies a separate position in the classification of IBPs and contribute to our understanding of interaction between macromolecules and ice and more generally, between macromolecules and inorganic crystals. Ice-binding proteins (IBPs) affect ice crystal growth by attaching to crystal faces. We present the effects on the growth of an ice single crystal caused by an ice-binding protein from the sea ice microalga Fragilariopsis cylindrus (fcIBP) that is characterized by the widespread domain of unknown function 3494 (DUF3494) and known to cause a moderate freezing point depression (below 1 °C). By the application of interferometry, bright-field microscopy, and fluorescence microscopy, we observed that the fcIBP attaches to the basal faces of ice crystals, thereby inhibiting their growth in the c direction and resulting in an increase in the effective supercooling with increasing fcIBP concentration. In addition, we observed that the fcIBP attaches to prism faces and inhibits their growth. In the event that the effective supercooling is small and crystals are faceted, this process causes an emergence of prism faces and suppresses crystal growth in the a direction. When the effective supercooling is large and ice crystals have developed into a dendritic shape, the suppression of prism face growth results in thinner dendrite branches, and growth in the a direction is accelerated due to enhanced latent heat dissipation. Our observations clearly indicate that the fcIBP occupies a separate position in the classification of IBPs due to the fact that it suppresses the growth of basal faces, despite its moderate freezing point depression.