Antifreeze protein


Antifreeze proteins 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.

Non-colligative properties

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. The unusual properties of AFPs are attributed to their selective affinity for specific crystalline ice forms and the resulting blockade of the ice-nucleation process.

Thermal hysteresis

AFPs create a difference between the melting point and freezing point 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.
Thermal hysteresis is easily measured in the lab with a nanolitre osmometer. Organisms differ in their values of thermal hysteresis. The maximum level of thermal hysteresis shown by fish AFP is approximately -3.5 °C. However, insect antifreeze proteins are 10–30 times more active than fish proteins. This difference probably reflects the lower temperatures encountered by insects on land. In contrast, aquatic organisms are exposed only to -1 to -2 °C below freezing. During the extreme winter months, the spruce budworm resists freezing at temperatures approaching -30 °C. The Alaskan beetle Upis ceramboides can survive in a temperature of -60 °C by using antifreeze agents that are not proteins.
The rate of cooling can influence the thermal hysteresis value of AFPs. Rapid cooling can substantially decrease the nonequilibrium freezing point, and hence the thermal hysteresis value. Consequently, organisms cannot necessarily adapt to their subzero environment if the temperature drops abruptly.

Freeze tolerance versus freeze avoidance

Species containing AFPs may be classified as
Freeze avoidant: These species are able to prevent their body fluids from freezing altogether. Generally, the AFP function may be overcome at extremely cold temperatures, leading to rapid ice growth and death.
Freeze tolerant: These species are able to survive body fluid freezing. Some freeze tolerant species are thought to use AFPs as cryoprotectants to prevent the damage of freezing, but not freezing altogether. The exact mechanism is still unknown. However, it is thought AFPs may inhibit recrystallization and stabilize cell membranes to prevent damage by ice. They may work in conjunction with protein ice nucleators to control the rate of ice propagation following freezing.

Diversity

There are many known nonhomologous types of AFPs.

Fish AFPs

Antifreeze glycoproteins or AFGPs are found in Antarctic notothenioids and northern cod. They are 2.6-3.3 kD. AFGPs evolved separately in notothenioids and northern cod. In notothenioids, the AFGP gene arose from an ancestral trypsinogen-like serine protease gene.
The classification of AFPs became more complicated when antifreeze proteins from plants were discovered. Plant AFPs are rather different from the other AFPs in the following aspects:
  1. They have much weaker thermal hysteresis activity when compared to other AFPs.
  2. Their physiological function is likely in inhibiting the recrystallization of ice rather than in the preventing ice formation.
  3. Most of them are evolved pathogenesis-related proteins, sometimes retaining antifungal properties.

    Insect AFPs

There are two types of insect antifreeze proteins, Tenebrio and Dendroides AFPs which are both in different insect families. They are similar to one another, both being hyperactive and consist of varying numbers of 12- or 13-mer repeats of approximately 8.3 to 12.5 kD. Throughout the length of the protein, at least every sixth residue is a cysteine. It has recently been discovered that even highly truncated insect AFP may exhibit ice structuring and thermal hysteresis activities.
Tenebrio or Type V AFPs are found in beetles, whereas Dendroides or Choristoneura fumiferana AFPs are found in some Lepidoptera.

Sea ice organism AFPs

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 and Antarctic inland ice bacteria, as well as in cold-tolerant fungi.
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 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.. EfcIBP has a typical AFP structure consisting of multiple beta-sheets and an alpha-helix. Also, all the ice-binding polar residues are at the same site of the protein.

Evolution

The remarkable diversity and distribution of AFPs suggest the different types evolved recently in response to sea level glaciation occurring 1-2 million years ago in the Northern hemisphere and 10-30 million years ago in Antarctica. This independent development of similar adaptations is referred to as convergent evolution. There are two reasons why many types of AFPs are able to carry out the same function despite their diversity:
  1. Although ice is uniformly composed of water molecules, it has many different surfaces exposed for binding. Different types of AFPs may interact with different surfaces.
  2. Although the five types of AFPs differ in their primary structure of amino acids, when each folds into a functioning protein, they may share similarities in their three-dimensional or tertiary structure that facilitates the same interactions with ice.

    Mechanisms of action

AFPs are thought to inhibit growth by an adsorption–inhibition mechanism. 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.

Binding to ice

Normally, ice crystals grown in solution only exhibit the basal and prism faces, and appear as round and flat discs. However, it appears the presence of AFPs exposes other faces. It now appears the ice surface 2021 is the preferred binding surface, at least for AFP type I. Through studies on type I AFP, ice and AFP were initially thought to interact through hydrogen bonding. 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.

Binding mechanism and antifreeze function

According to the structure and function study on the antifreeze protein from Pseudopleuronectes americanus, 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.
The above mechanism can be used to elucidate the structure-function relationship of other antifreeze proteins with the following two common features:
  1. recurrence of a Thr residue in an 11-amino-acid period along the sequence concerned, and
  2. a high percentage of an Ala residue component therein.

    History

In the 1950s, Norwegian scientist Scholander set out to explain how Arctic fish can survive in water colder than the freezing point of their blood. His experiments led him to believe there was “antifreeze” in the blood of Arctic fish. Then in the late 1960s, animal biologist Arthur DeVries was able to isolate the antifreeze protein through his investigation of Antarctic fish. These proteins were later called antifreeze glycoproteins or antifreeze glycopeptides to distinguish them from newly discovered nonglycoprotein biological antifreeze agents. DeVries worked with Robert Feeney to characterize the chemical and physical properties of antifreeze proteins. In 1992, Griffith et al. documented their discovery of AFP in winter rye leaves. Around the same time, Urrutia, Duman and Knight documented thermal hysteresis protein in angiosperms. 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.

Name change

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.

Commercial and medical applications

Numerous fields would be able to benefit from the protection of tissue damage by freezing. Businesses are currently investigating the use of these proteins in:
Unilever has obtained UK approval to use a genetically modified yeast to produce antifreeze proteins from fish, for use in ice cream production.

Recent news

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 who believe that antifreeze proteins may cause inflammation. Intake of AFPs in diet is likely substantial in most northerly and temperate regions already. Given the known historic consumption of AFPs, it is safe to conclude their functional properties do not impart any toxicologic or allergenic effects in humans.
As well, the transgenic process of ISP production is widely used in society already. Insulin and rennet are produced using this technology. The process does not impact the product; it merely makes production more efficient and prevents the death of fish which would otherwise be killed to extract the protein.
Currently, Unilever incorporates AFPs into some of its American products, including some Popsicle ice pops and a new line of Breyers Light Double Churned ice cream bars. In ice cream, AFPs allow the production of very creamy, dense, reduced fat ice cream with fewer additives. 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.
A 2010 study demonstrated the stability of superheated water ice crystals in an AFP solution, showing that while the proteins can inhibit freezing, they can also inhibit melting.