E Pluribus Unum: Functional Aggregation Enables Biological Ice Nucleation

Biological ice nucleation plays a key role in the survival and adaptation of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation of ice at temperatures above -10 oC. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 oC. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here we combine ice-nucleation measurements, physicochemical characterization, numerical modeling and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits which assemble into ice-nucleating complexes that contain more than 100 subunits and have an ice-binding area of at least 250 nm2. The potency of the INs is retained even when only the smaller subunits are initially present, suggesting robust pathways for their functional assembly in solution. We conclude that the use of small protein building blocks to build large IN assemblies is the common strategy among organisms to create potent biological INs.