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Class of peptides which help cells survive freezing conditions

Antifreeze proteins (AFPs) or ice structuring proteins refer to a class of polypeptides produced by certain animals, plants, fungi and bacteria that permit their survival in temperatures below the freezing point of water. AFPs bind to small ice crystals to inhibit the growth and recrystallization of ice that would otherwise be fatal. There is also increasing evidence that AFPs interact with mammalian cell membranes to protect them from cold damage. This work suggests the involvement of AFPs in cold acclimatization.

Unlike the widely used automotive antifreeze, ethylene glycol, AFPs do not lower freezing point in proportion to concentration. Rather, they work in a noncolligative manner. This phenomenon allows them to act as an antifreeze at concentrations 1/300th to 1/500th of those of other dissolved solutes. Their low concentration minimizes their effect on osmotic pressure.

AFPs create a difference between the melting point and freezing point (busting temperature of AFP bound ice crystal) known as thermal hysteresis. The addition of AFPs at the interface between solid ice and liquid water inhibits the thermodynamically favored growth of the ice crystal. Ice growth is kinetically inhibited by the AFPs covering the water accessible surfaces of ice. They may work in conjunction with ice nucleating proteins (INPs) to control the rate of ice propagation following freezing. AFGPs evolved separately in notothenioids and northern cod. In notothenioids, the AFGP gene arose from an ancestral trypsinogen like serine protease gene. Type I AFP is found in winter flounder, longhorn sculpin and shorthorn sculpin. It is the best documented AFP because it was the first to have its three dimensional structure determined. Type I AFP consists of a single, long, amphipathic alpha helix, about 3.3 4.5 kD in size. There are three faces to the 3D structure: the hydrophobic, hydrophilic, and Thr Asx face. The ability is partially derived from its many repeats of the Type I ice binding site. Type II AFPs (e. g. ) are found in sea raven, smelt and herring. They are cysteine rich globular proteins containing five disulfide bonds. Type II AFPs likely evolved from calcium dependent (c type) lectins. Sea ravens, smelt, and herring are quite divergent lineages of teleost. If the AFP gene were present in the most recent common ancestor of these lineages, it is peculiar that the gene is scattered throughout those lineages, present in some orders and absent in others. It has been suggested that lateral gene transfer could be attributed to this discrepancy, such that the smelt acquired the type II AFP gene from the herring. Type III AFPs are found in Antarctic eelpout. They exhibit similar overall hydrophobicity at ice binding surfaces to type I AFPs. They are approximately 6kD in size. Type IV AFPs are found in longhorn sculpins. They are alpha helical proteins rich in glutamate and glutamine. This protein is approximately 12KDa in size and consists of a 4 helix bundle. Plant AFPs are rather different from the other AFPs in the following aspects:

They have much weaker thermal hysteresis activity when compared to other AFPs. Their physiological function is likely in inhibiting the recrystallization of ice rather than in preventing ice formation. Other beetles (genus Rhagium) have longer repeats without internal disulfide bonds that form a compressed beta solenoid (beta sandwich) with four rows of threonine residus, and this AFP is structurally similar to that modelled for the non homologous AFP from the pale beauty moth. In contrast, the AFP from the spruce budworm moth is a solenoid that superficially resembles the Tenebrio protein, with a similar ice binding surface, but it has a triangular cross section, with longer repeats that lack the internal disulfide bonds. The AFP from midges is structurally similar to those from Tenebrio and Dendroides, but the disulfide braced beta solenoid is formed from shorter 10 amino acids repeats, and instead of threonine, the ice binding surface consists of a single row of tyrosine residues. Springtails (Collembola) are not insects, but like insects, they are arthropods with six legs. A species found in Canada, which is often called a "snow flea", produces hyperactive AFPs. Other insects, such as an Alaskan beetle, produce hyperactive antifreezes that are even less similar, as they are polymers of sugars (xylomannan) rather than polymers of amino acids (proteins). Taken together, this suggests that most of the AFPs and antifreezes arose after the lineages that gave rise to these various insects diverged. The similarities they do share are the result of convergent evolution.

Many microorganisms living in sea ice possess AFPs that belong to a single family. The diatoms Fragilariopsis cylindrus and F. curta play a key role in polar sea ice communities, dominating the assemblages of both platelet layer and within pack ice. AFPs are widespread in these species, and the presence of AFP genes as a multigene family indicates the importance of this group for the genus Fragilariopsis. AFPs identified in F. cylindrus belong to an AFP family which is represented in different taxa and can be found in other organisms related to sea ice (Colwellia spp., Navicula glaciei, Chaetoceros neogracile and Stephos longipes and Leucosporidium antarcticum) and Antarctic inland ice bacteria (Flavobacteriaceae), as well as in cold tolerant fungi (Typhula ishikariensis, Lentinula edodes and Flammulina populicola). Several structures for sea ice AFPs have been solved. This family of proteins fold into a beta helix that form a flat ice binding surface. Unlike the other AFPs, there is not a singular sequence motif for the ice binding site. AFP found from the metagenome of the ciliate Euplotes focardii and psychrophilic bacteria has an efficient ice re crystallization inhibition ability. 1 μM of Euplotes focardii consortium ice binding protein (EfcIBP) is enough for the total inhibition of ice re crystallization in –7.4 °C temperature. This ice recrystallization inhibition ability helps bacteria to tolerate ice rather than preventing the formation of ice. EfcIBP produces also thermal hysteresis gap, but this ability is not as efficient as the ice recrystallization inhibition ability. EfcIBP helps to protect both purified proteins and whole bacterial cells in freezing temperatures. Green fluorescent protein is functional after several cycles of freezing and melting when incubated with EfcIBP. Escherichia coli survives longer periods in 0 °C temperature when the efcIBP gene was inserted to E. coli genome. The cooling of Antarctic imposed from this current caused a mass extinction of teleost species that were unable to withstand freezing temperatures. Notothenioids species with the antifreeze gylcoprotein were able to survive the glaciation event and diversify into new niches. Although the two fish orders have similar antifreeze proteins, cod species contain arginine in AFG, while Antarctic notothenioid do not. Previous research has found various enhancers of this bettles' antifreeze protein including a thaumatin like protein and polycarboxylates. Modifications of DAFP 1 with the arginine specific reagent resulted in the partial and complete loss of thermal hysteresis in DAFP 1, indicating that arginine plays a crucial role in enhancing its ability. TH activity of DAFP 4 was not affected by pH unless the there was a low solute concentration (pH 1) in which TH decreased. The direction of transfer is known to be from herring to smelt as herring have 8 times the copies of AFP gene as smelt (1) and the segments of the gene in smelt house transposable elements which are otherwise characteristic of and common in herring but not found in other fishes. Antifreeze glycoprotein activity has been observed across several ray finned species including eelpouts, sculpins, and cod species. Fish species that possess the antifreeze glycoprotein express different levels of protein activity. Polar cod (Boreogadus saida) exhibit similar protein activity and properties to the Antarctic species, T. borchgrevinki. Horizontal gene transfer is responsible for the presence of ice antifreeze proteins in two sea diatom species, F. cylindrus and F. curta. They adsorb to nonbasal planes of ice, inhibiting thermodynamically favored ice growth. The presence of a flat, rigid surface in some AFPs seems to facilitate its interaction with ice via Van der Waals force surface complementarity.

Normally, ice crystals grown in solution only exhibit the basal (0001) and prism faces (1010), and appear as round and flat discs. Through studies on type I AFP, ice and AFP were initially thought to interact through hydrogen bonding (Raymond and DeVries, 1977). However, when parts of the protein thought to facilitate this hydrogen bonding were mutated, the hypothesized decrease in antifreeze activity was not observed. Recent data suggest hydrophobic interactions could be the main contributor. It is difficult to discern the exact mechanism of binding because of the complex water ice interface. Currently, attempts to uncover the precise mechanism are being made through use of molecular modelling programs (molecular dynamics or the Monte Carlo method). the antifreeze mechanism of the type I AFP molecule was shown to be due to the binding to an ice nucleation structure in a zipper like fashion through hydrogen bonding of the hydroxyl groups of its four Thr residues to the oxygens along the direction in ice lattice, subsequently stopping or retarding the growth of ice pyramidal planes so as to depress the freeze point. These proteins were later called antifreeze glycoproteins (AFGPs) or antifreeze glycopeptides to distinguish them from newly discovered nonglycoprotein biological antifreeze agents (AFPs). DeVries worked with Robert Feeney (1970) to characterize the chemical and physical properties of antifreeze proteins. In 1992, Griffith et al. documented their discovery of AFP in winter rye leaves. The next year, Duman and Olsen noted AFPs had also been discovered in over 23 species of angiosperms, including ones eaten by humans. They reported their presence in fungi and bacteria as well.

Recent attempts have been made to relabel antifreeze proteins as ice structuring proteins to more accurately represent their function and to dispose of any assumed negative relation between AFPs and automotive antifreeze, ethylene glycol. These two things are completely separate entities, and show loose similarity only in their function.

One recent, successful business endeavor has been the introduction of AFPs into ice cream and yogurt products. This ingredient, labelled ice structuring protein, has been approved by the Food and Drug Administration. The proteins are isolated from fish and replicated, on a larger scale, in genetically modified yeast. There is concern from organizations opposed to genetically modified organisms (GMOs) who believe that antifreeze proteins may cause inflammation. Intake of AFPs in diet is likely substantial in most northerly and temperate regions already. They control ice crystal growth brought on by thawing on the loading dock or kitchen table, which reduces texture quality. In November 2009, the Proceedings of the National Academy of Sciences published the discovery of a molecule in an Alaskan beetle that behaves like AFPs, but is composed of saccharides and fatty acids. In 2021, EPFL and Warwick scientists have found an artificial imitation of antifreeze proteins.