Antifreeze proteins (AFPs) are specific proteins that are able to lower the freezing point of aqueous solutions relative to the melting point. Hyperactive AFPs, identified in insects, have an especially high ability to depress the freezing point by far exceeding the abilities of other AFPs. In previous studies, we postulated that the activity of AFPs can be attributed to two distinct molecular mechanisms: ( i ) short-range direct interaction of the protein surface with the growing ice face and ( ii ) long-range interaction by protein-induced water dynamics extending up to 20 Å from the protein surface. In the present paper, we combine terahertz spectroscopy and molecular simulations to prove that long-range protein–water interactions make essential contributions to the high antifreeze activity of insect AFPs from the beetle Dendroides canadensis . We also support our hypothesis by studying the effect of the addition of the osmolyte sodium citrate.

Significance: Controlling ice crystal growth is a grand scientific challenge with major technological ramifications. Some cold-adapted organisms such as fish and arthropods are protected against ice growth by producing antifreeze proteins (AFPs). These AFPs adsorb onto embryonic ice crystals, thereby inhibiting their growth. On a macroscopic level, this is evidenced by ice recrystallization inhibition (IRI) and thermal hysteresis (TH) activity. Our research demonstrates the absence of a clear correlation between TH and IRI activities, and underlines the importance of the extent of supercooling and annealing time on the efficacy of AFPs in blocking ice growth. We emphasize that successful application of AFPs and synthetic analogues in innovative strategies for cryoprotection, cryopreservation, and antiicing technologies requires optimization tailored toward the specific purpose.

Significance: Antifreeze proteins (AFPs) enable the survival of various organisms in freezing or subfreezing habitats by preventing the macroscopic growth of ice crystals. Understanding how AFPs recognize and bind ice crystals is the most important step to unravel their working mechanism. Using surface-specific sum frequency generation spectroscopy, we were able to directly probe the ice-binding site of the protein and discovered that, already at room temperature and in an aqueous solution, the antifreeze proteins arrange water molecules into an ice-like array, which they then use to bind to ice crystals.

A new kid in town: 3D Raman intensity images of a HT29 colon cancer cell incubated with an aqueous solution of the CO‐releasing metal–carbonyl complex 1 show that the compound penetrates the cellular membrane and is localized mainly in the nuclear membrane and the nucleolus. The CO vibration of 1 serves as an intrinsic spectroscopic label.

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.