Bacterial ice-nucleating proteins (INPs) promote heterogeneous ice nucleation more efficiently than any other material. The details of their working mechanism remain elusive, but their high activity has been shown to involve the formation of functional INP aggregates. Here we reveal the importance of electrostatic interactions for the activity of INPs from the bacterium Pseudomonas syringae by combining a high-throughput ice nucleation assay with surface-specific sum-frequency generation spectroscopy. We determined the charge state of nonviable P. syringae as a function of pH by monitoring the degree of alignment of the interfacial water molecules and the corresponding ice nucleation activity. The net charge correlates with the ice nucleation activity of the INP aggregates, which is minimal at the isoelectric point. In contrast, the activity of INP monomers is less affected by pH changes. We conclude that electrostatic interactions play an essential role in the formation of the highly efficient functionally aligned INP aggregates, providing a mechanism for promoting aggregation under conditions of stress that prompt the bacteria to nucleate ice.

The large-scale production and ecotoxicity of urea make its removal from wastewater a health and environmental challenge. Whereas the industrial removal of urea relies on hydrolysis at elevated temperatures and high pressure, nature solves the urea disposal problem with the enzyme urease under ambient conditions. We show that CeO2-x nanorods (NRs) act as the first and efficient green urease mimic that catalyzes the hydrolysis of urea under ambient conditions with an activity (kcat = 9.58 × 101 s-1) about one order of magnitude lower than that of the native jack bean urease. The surface properties of CeO2-x NRs were probed by varying the Ce4+/Ce3+ ratio through La doping. Although La substitution increased the number of surface defects, the reduced number of Ce4+ sites with higher Lewis acidity led to a slight decrease of their catalytic activity. CeO2-x NRs are stable against pH changes and even to the presence of transition metal ions like Cu2+, one of the strongest urease inhibitors. The low costs and environmental compatibility make CeO2-x NRs a green urease substitute that may be applied in polymer membranes for water processing or filters for the waste water reclamation. The biomimicry approach allows the application of CeO2-x NRs as functional enzyme mimics where the use of native or recombinant enzyme is hampered because of its costs or operational stability.

Ice-nucleating proteins (INPs) found in bacteria are the most effective ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C. Although their function has been known for decades, the underlying mechanism is still under debate. Here, we show that INPs from Pseudomonas syringae in aqueous solution exhibit a defined solution structure and show no significant conformational changes upon cooling. In contrast, irreversible structural changes are observed upon heating to temperatures exceeding ∼55 °C, leading to a loss of the ice-nucleation activity. Sum-frequency generation (SFG) spectroscopy reveals that active and heat-inactivated INPs impose similar structural ordering of interfacial water molecules upon cooling. Our results demonstrate that increased water ordering is not sufficient to explain INPs' high ice-nucleation activity and confirm that intact three-dimensional protein structures are critical for bacterial ice nucleation, supporting a mechanism that depends on the INPs' supramolecular interactions.

Bacterial ice nucleators (INs) are among the most effective ice nucleators known and are relevant for freezing processes in agriculture, the atmosphere, and the biosphere. Their ability to facilitate ice formation is due to specialized ice-nucleating proteins (INPs) anchored to the outer bacterial cell membrane, enabling the crystallization of water at temperatures up to −2 °C. In this Perspective, we highlight the importance of functional aggregation of INPs for the exceptionally high ice nucleation activity of bacterial ice nucleators. We emphasize that the bacterial cell membrane, as well as environmental conditions, is crucial for a precise functional INP aggregation. Interdisciplinary approaches combining high-throughput droplet freezing assays with advanced physicochemical tools and protein biochemistry are needed to link changes in protein structure or protein–water interactions with changes on the functional level.

Cold-adapted organisms use antifreeze proteins (AFPs) or ice-nucleating proteins (INPs) for the survival in freezing habitats. AFPs have been reported to be able to inhibit the activity of INPs, a property that would be of great physiological relevance. The generality of this effect is not understood, and for the few known examples of INP inhibition by AFPs, the molecular mechanisms remain unclear. Here, we report a comprehensive evaluation of the effects of five different AFPs on the activity of bacterial ice nucleators using a high-throughput ice nucleation assay. We find that bacterial INPs are inhibited by certain AFPs, while others show no effect. Thus, the ability to inhibit the activity of INPs is not an intrinsic property of AFPs, and the interactions of INPs and different AFPs proceed through protein-specific rather than universal molecular mechanisms.