Protein engineering of antifreeze proteins reveals that their activity scales with the area of the ice‐binding site

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

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Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period

Scholl

Holmstrup

Graham

et al. 2023

Sci Rep

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Long-range, water-mediated interaction between a moderately active antifreeze protein molecule and the surface of ice

Grabowska,

Kuffel,

Zielkiewicz

2024

The Journal of Chemical Physics

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Using molecular dynamics simulations, we show that a molecule of moderately active antifreeze protein (type III AFP, QAE HPLC-12 isoform) is able to interact with ice in an indirect manner. This interaction occurs between the ice binding site (IBS) of the AFP III molecule and the surface of ice, and it is mediated by liquid water, which separates these surfaces. As a result, the AFP III molecule positions itself at a specific orientation and distance relative to the surface of ice, which enables the effective binding (via hydrogen bonds) of the molecule with the nascent ice surface. Our results show that the final adsorption of the AFP III molecule on the surface of ice is not achieved by chaotic diffusion movements, but it is preceded by a remote, water-mediated interaction between the IBS and the surface of ice. The key factor that determines the existence of this interaction is the ability of water molecules to spontaneously form large, high-volume aggregates that can be anchored to both the IBS of the AFP molecule and the surface of ice. The results presented in this work for AFP III are in full agreement with the ones obtained by us previously for hyperactive CfAFP, which indicates that the mechanism of the remote interaction of these molecules with ice remains unchanged despite significant differences in the molecular structure of their ice binding sites. For that reason, we can expect that also other types of AFPs interact with the ice surface according to an analogous mechanism.

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On the engulfment of antifreeze proteins by ice

Thosar,

Cai,

Marks

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Antifreeze proteins (AFPs) are remarkable biomolecules that suppress ice formation at trace concentrations. To inhibit ice growth, AFPs must not only bind to ice crystals, but also resist engulfment by ice. The highest supercooling, Δ T ∗ , for which AFPs are able to resist engulfment is widely believed to scale as the inverse of the separation, L , between bound AFPs, whereas its dependence on the molecular characteristics of the AFP remains poorly understood. By using specialized molecular simulations and interfacial thermodynamics, here, we show that in contrast with conventional wisdom, Δ T ∗ scales as L − 2 and not as L − 1 . We further show that Δ T ∗ is proportional to AFP size and that diverse naturally occurring AFPs are optimal at resisting engulfment by ice. By facilitating the development of AFP structure–function relationships, we hope that our findings will pave the way for the rational design of AFPs.

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Protein engineering of antifreeze proteins reveals that their activity scales with the area of the ice‐binding site

Cited by 3 publications

References 31 publications

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period

Long-range, water-mediated interaction between a moderately active antifreeze protein molecule and the surface of ice

On the engulfment of antifreeze proteins by ice

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