Antifreeze protein is currently a hot topic of interest.The function of the antifreeze protein is to lower the freezing temperature and to restrict the ice formation and change the ice nature by suppressing the growth of ice nuclei. It also delays the recrystallization on frozen storage. It is involved in different kind of functions like increase storage life of fruits, make the animals temperature tolerant, prevent crystal formation so, improve yield quality.Antifreeze proteins are used to tackle the problem and store food products in frozen form without loss of any texture. Antifreeze proteins are used in the food sector in products like ice cream, frozen fish and meat, and frozen dough in order to ensure the uniform texture in products.

1. Antifreeze Protein

2. • Antifreeze proteins (AFPs) are a group of proteins that
protect organisms living in extremely cold climates from
deep freezing temperatures. These are observed in
vertebrates, invertebrates, plants, bacteria, and fungi.
• These proteins are able to utilize their unique structures
that hinder freezing by binding to and preventing young
ice crystals from growing.
• AFPs can effectively protect organisms from damage
during freezing conditions by a process termed thermal
hysteresis. This phenomenon is the presence of a
hysteresis gap, that is better explained by the lowering of
the freezing point non-colligatively, leaving the melting
point unchanged.
What are AFP’s?

3. History
In the 1950s, Norwegian scientist Scholander explained, 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 De-Vries was able to
isolate the antifreeze protein Antarctic fish. In 1970, De-Vries 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 (1992)
documented thermal hysteresis protein in angiosperms.
The next year, Duman and Olsen noted AFPs had also been
discovered in over 23 species of angiosperm including ones eaten
by humans and also present in fungi and bacteria as well.

4. Evolution:
The remarkable diversity of AFPs suggests 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.
There are two reasons why many protein types carry same function:
– Even ice is uniformly composed by water molecules but still have
different binding sites so, different AFPs react with sites.
– The five types of AFPs differ in their primary structure of A.A when
folds into a functioning proteins, may share similarities in their 3-D
or tertiary structure, provides the same binding with ice.
Name change:
Recently antifreeze proteins are re-labeled as ice structuring
proteins to show their accurate function and to dispose of any
relation between AFPs and automotive antifreeze(ethylene glycol)
which are completely separate entities, and show loose similarity
only in their function.

5. Properties
• Thermal Hysteresis:
It results from the lowering of the apparent freezing temperature
without actually affecting the melting point. This causes AFPs to be
200 to 300 times more effective at freezing point depressions than in
ideal solutions.
• Pathogenesis:
Most of the antifreeze proteins sequenced has shown that they are
homologous to pathogenesis-related proteins. They are released
into the apoplast in response to a pathogenic infection.
• Freeze tolerance versus freeze avoidance:
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.

6. Protein Species Structural
type
Protein
homology
Ice binding
site
Fish type I AFP
Right eye
flounder, sculpins
single, long,
amphipathic
and alpha helix
undetected
Thr and residues
associated
Fish type II AFP
sea raven,
rainbow smelt,
Atlantic herring
globular; smelt
and herring are
Ca2+ dependent
galactose-
binding; +- C-
type lectins in
herring
with it
corresponds to
Ca2+ dependent
binding site
Fish type III
AFP eel pouts such as
ocean pout
globular with
one flattened
surface
undetected
residues on and
flanking flat
surface
Fish type IV
AFP
longhorn sculpin antiparallel helix
bundle
low-density
lipoprotein
receptor-
binding domain
E3
unknown

7. Mechanism of action
• Crystallization involves two major steps: nucleation,
which is the formation of a stable crystal nucleus and
propagation of ice crystals by the growth of the nucleus.
• Nucleation generally occurs around a foreign molecule
and re-crystallization of ice occurs when the temperature
fluctuates.
• These are more potent to cause physical damage to
tissues and cells.
• AFPs are thought to inhibit growth by an adsorption
inhibition mechanism. They adsorb to non-basal
planes of ice, inhibiting thermodynamically-favored ice
growth.

8. • The presence of a flat, rigid surface in some
AFPs seems to facilitate its interaction with ice
via Van-der-Waals force surface
complementarity.
Mechanism of action

9. Binding to Ice
• Normally, ice crystals grown in solution only exhibit the
basal (0001) and prism faces (1010), 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.

10. • 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.

11. • Currently, attempts to uncover the precise mechanism
are being made through use of molecular modelling
programs.

12. Antifreeze Function
Structural and function study on
the antifreeze protein
from Pseudopleuronectes
americanus the antifreeze
mechanism of the type-I AFP
molecule was shown 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 oxygen along
the [0112] direction in ice lattice,
subsequently stop the growth of
ice pyramidal planes to depress
the freezing point.

13. • The above mechanism can be used to elucidate
the structure-function relationship of other
antifreeze proteins with the following two
common features:
Antifreeze Function
recurrence of a Thr residue (or any other polar amino acid
residue whose side-chain can form a hydrogen bond with water)
in an 11-amino-acid period along the sequence concerned, and
a high percentage of an Ala residue component therein.
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