Cite this: RSC Advances, 2013, 3,14199

Natural macromolecular antifreeze agents to syntheticantifreeze agents

Received 8th January 2013,Accepted 26th April 2013

DOI: 10.1039/c3ra00081h

www.rsc.org/advances

V. Haridas* and Sarala Naik

Many living systems native to the Arctic and Antarctic regions express antifreeze proteins (AFPs) or

antifreeze glycoproteins (AFGPs) that recognize and bind to specific faces of ice crystals, thereby inhibiting

ice growth. The non-colligative freezing point depression induced by these proteins results primarily from

their unique chemical structures. This review describes the various classes of AF(G)Ps, their structural

hierarchy, their mechanisms of action, and novel synthetic antifreeze compounds. The mechanism of

action of AF(G)Ps displays a high degree of precision present in natural systems. The varied chemical

structures of AFPs with similar antifreeze activities suggest convergent evolution. Structural studies of AFPs

from insects, plants and bacteria have revealed unusual beta helical structures. A variety of AF(G)P analogs

have been synthesized and have revealed the mechanisms underlying the action of AF(G)Ps. The utility of

AF(G)Ps and their analogs in cryopreservation, cryosurgery, and the food industry motivates the

development of new artificial antifreeze agents.

I. Introduction

Living systems adopt a variety of strategies to cope withparticular environmental conditions. Adaptation to an envir-onment is an evolutionary process involving changes at the

Department of chemistry, Indian Institute of Technology Delhi (IITD), Hauz Khas,

New Delhi, 110016, India. E-mail: h_haridas@hotmail.com; Tel: 01126591380

Dr V. Haridas is an AssociateProfessor in the Department ofChemistry at the Indian Institute ofTechnology Delhi (IITD), India. Hefinished his Ph.D under the guidanceof Prof. D. Ranganathan (NationalInstitute for Interdisciplinary Scienceand Technology, India). He did hispost doctoral studies with Prof. R. M.Ghadiri (The Scripps ResearchInstitute, USA) as a Skaggs Fellowand with Prof. Herbert Waldmann(Max Planck Institute, Germany) asan Alexander Humboldt and Max

Planck Fellow. His research group at IIT Delhi is working in the area ofChemical Biology.

Dr Sarala Naik was born inSundargarh, Orissa, India (1978).She did her M.Sc in OrganicSynthesis and Biochemistry atSambalpur University, Orissa.She obtained her Ph.D degree insynthetic organic chemistry underthe guidance of Prof. Bhisma K.Patel (Indian Institute ofTechnology Guwahati, India) inthe year 2007. She worked as aresearch associate under thesupervision of Dr V. Haridas indendrimer chemistry in the Indian

Institute of Technology Delhi, India (2007–2008) and as postdoctoral fellow (2008–2009) in synthesis and photophysical studiesof fluorophores under the co-guidance of Dr M. S. T. Gonçalves andDr P. J. G. Coutinho in the University of Minho, Braga, Gualtar,Portugal with a FCT fellowship grant. Currently she is working inthe IITD as the principal investigator for the DST-SERC FAST Trackproject grant. Her research interests include synthetic organicchemistry, green chemistry, bioorganic chemistry, fluorophorechemistry with evaluation ofbiocompatibilityy of long wavelengthbenzo[a]phenoxazine fluorophores.

V. Haridas Sarala Naik

RSC Advances

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molecular level, which manifest as physical changes.1,2

Temperature is a significant environmental factor responsiblefor the distribution of living organisms. Although prokaryotescan survive under extreme thermal conditions, the survival ofeukaryotic organisms under such conditions is challenging. Atsubzero temperatures, ice crystals can enter the body of anorganism by ingestion and circulate, leading to further crystalgrowth that causes cell and tissue damage and subsequentdeath. Low temperatures reduce the rates of metabolic andbiochemical reactions and physiological processes; a largeramount of energy must be expended by the organism foractivity, growth, and reproduction.3 Notwithstanding thechallenges posed by low temperatures, the survival of a largenumber of aquatic invertebrates and vertebrates in polarenvironments attests to the fact that these organisms haveadapted over evolutionary time.4 The survival of fish underextremely cold conditions in the polar regions suggests thatevolution played a significant role in adaptation and thatantifreeze (glyco)proteins (AF(G)Ps) evolved to mitigate thethreat posed by freezing icy seawater.5

Fig. 1 Flow chart showing the classification of antifreeze biomolecules.

Table 1 Salient features of various natural antifreeze proteins

Types of AFP Structure Mol. wt. Special Features Evolutionary Precursors

Type I Single a-helix 3.3–4.5 kDa [Thr-(X)2-Y-(X)7]-X = Nonpolaralanine residue . 60%,Y = polar residue

Unknown

Amphipathic a-helix,11-residue repeat

Type II Globular 11–24 kDa Cysteine (8%), globular, highamount of hydrophilic aminoacids, five disulfidebonds, non-repetitive

C-type lectins

Type III Globular,short b-strands

6.5 kDa Non-repetitive Sialic acid synthaseC-terminus

Type IV a-helical bundle 12 kDa Helical bundles, glutaminerich, 22-residue repeats

Apolipoprotein

AFGP Extended 3-fold helix 2.6–33 kDa (Ala-Ala-Thr-carbohydrate)n Trypsinogen like serine proteaseAlanine . 60%, threonine . 30%,amphipathic extended polyprolinetype II helix, 3-residue repeats

Beetle AFP (TmAFP) Right-handed b-helix 8.4 kDa 12- and 13-amino acid residuerepeats -CTXSXXCXXAXT-,X = any amino acid, TCT motifsform b-sheet-putative ice bindingsurface, eight disulfide bonds

Unknown

Moth AFP (sbwAFP) Left-handed b-helix 9 kDa 15-amino acid loops UnknownCys, Thr, Ser-richRepetitive TXT

Hyperactive type I AFP Long a-helix 16.7 kDa (monomer) Dimeric, side by sidearrangement of two a-helices

Unknown32.3 kDa (dimer)

Hyperactive bacterialAFP, MpAFP_RIV

b-helix 34 kDa Ca2+-dependent RTX (Repeats-in-Toxin)family of proteins

Snow flea AFP (sfAFP) PPII helices 6.5 kDa (small isoform) Gly and Ala-rich Unknown15.7 kDa(large isoform)

Four Cys forming disulfide bonds(in 6.5 kDa protein), two Cysforming disulfide bonds(in 15.7 kDa protein)

Longhorn beetleAFP (RiAFP)

b-helix 13 kDa TXTXTXT motifs, X = Ala or Thr Unknown

Plant AFP (LpAFP) b-helix 29 kDa Multiple hydrophilic ice bindingdomain, binds to the basaland primary-prism planes

Unknown

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The existence of AF(G)Ps provides an evolutionary example ofthe development of a new functional property (antifreeze) in theface of strong environmental pressure, and AF(G)Ps serve as anexample of adaptive molecular evolution.6 The evolutionaryorigins of half of the known AF(G)Ps have not yet beendetermined. The elucidation of plausible evolutionary originsfor the AF(G)Ps could broaden our understanding of molecularevolution under an environmental threat. Genetic analysisindicated that Antarctic notothenioid AFGP genes originatedfrom pancreatic trypsinogen.7 Arctic cod antifreeze glycoprotein(AFGP) genes have no sequence similarities with the trypsinogengene, indicating that trypsinogen was not the progenitor. Theseparate genomic origin and, thus, the separate ancestry of thesetwo polar fishes, which both produce antifreeze glycoproteins,indicate that the AFGPs evolved separately and arrived at asimilar function through convergent evolution.8 AFPs have a veryrecent evolutionary history9 and multiple evolutionary origins.10

Diversity among AF(G)Ps is a consequence of independentevolutionary origin and the surface heterogeneities of ice.11

Polar fishes possess certain biomacromolecular AF(G)Ps intheir blood serum that prevent freezing (ice crystal formation)and depress the serum freezing point below the colligativefreezing point of the body fluids.12 AF(G)Ps are present in theblood sera and body fluids of polar fishes,13 cold-tolerantinsects,14 Antarctic bacteria,15 and plants.16 Polar fishes produceAFPs and AFGPs that circulate in the blood at levels as high as 40mg mL21.17 AF(G)Ps in fish have been most extensively studied,which has been addressed in many reviews.18 These proteinslower the freezing point of a solution without making anappreciable change to the melting point or osmotic pressure ofthat solution. Interestingly, some of the AF(G)Ps do affect themelting point of ice.19 The difference between the freezing pointand the melting point temperatures is called thermal hysteresis(TH). TH was first reported by Ramsay in the context of themealworm Tenebrio molitor (Tm).20 A few years later, it wasobserved in the body fluids of polar fishes, thus elevating thephenomenon to a broader area of research.21,22

Fish AFPs yield a hysteresis of the order of 0.9 uC, whereas insectAFPs yield a hysteresis of >5 uC. The relationship between the THactivity and the AFP concentration is nonlinear and hyperbolic.23

The mechanism underlying the freezing point depression inducedby an AFP has presented a mechanistic and thermodynamicpuzzle. Here, we summarize the sources, types of natural antifreezemolecules, synthetic AF(G)Ps and also review the mechanistichypothesis underlying the antifreeze behavior of AF(G)Ps.

Researchers have extensively studied the structures of AF(G)Psto understand the mechanisms by which these proteins inhibitice crystal growth and depress the freezing point of body fluids,thereby making survival feasible under extremely cold condi-tions.24 The first antifreeze activity in marine fishes was reportedby Scholander in 1957, but the isolation and purification of anantifreeze compound from Antarctic notothenioid fish was notachieved until the year 1969 by DeVries.4,5 It comprised a series ofglycoproteins and, hence, is referred to as an antifreezeglycoprotein (AFGP).5 AFGPs are prevalent in the Arctic,Antarctic and among North Atlantic cod. Investigations by

DeVries and colleagues revealed the surprising role of thesebiomacromolecules in the blood of polar fishes.25 Biologicalantifreeze molecules are broadly categorized as either AFGPs orAFPs. The order of discovery, composition, and structuraldiversity of the fish AFPs led to their classification as type I–IVAFPs and AFGPs (Fig. 1, Table 1). Each type of AFP differs from theothers in the primary, secondary, and tertiary structures.

II. Types of AFPs

II A. Type I AFPs

Type I AFPs are the simplest macromolecular AFPs character-ized to date and are found in the winter flounder, yellowtail

Fig. 2 HPLC profiles of type I AFPs from (a) winter flounder,28 (b) shorthornsculpin,29 and (c) primary structures of the alanine rich type I AFP.

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flounder, Alaskan plaice, and shorthorn sculpin. AFP andAFGP types may be expressed as a mixture of several isoforms,and cooperative effects among the isoforms play a major rolein the antifreeze activity.26,27 The reverse phase HPLC profilesof AFPs isolated from the serum of winter flounder revealedeight components,28 HPLC-1 to HPLC-8 (Fig. 2a), among whichthe HPLC-6 isoform is the most extensively studied. The HPLC-6 isoform of the winter flounder AFP has an a-helical structurewith 37 amino acids, 23 of which are alanine (62%).29 TheHPLC-6 isoform consists of three sequence repeats of 11amino acids with the [Thr-(X)2-Y-(X)7]-motif, where X indicatesa nonpolar amino acid and Y indicates a polar residue. Thepresence of alanine residues in a higher percentage favors thealpha helical conformations in these proteins.30,31

Yellowtail flounder AFP comprises of 48 amino acids(Fig. 2).32 The shorthorn sculpin (SS) AFP assumes two majorisoforms, SS-3 and SS-8, containing 33 and 45 amino acids,respectively (Fig. 2b and 2c).33 The winter flounder AFP andshorthorn sculpin AFP are structurally homologous andcontain a high alanine content. The helical content of theHPLC-6 and HPLC-8 isoforms is 85%, whereas the helicalcontent of the SS-8 isoform is 73% and that of the SS-3 isoformis 45%. The SS-3 isoform is the smallest naturally occurringtype I AFP. It includes a helix-breaking proline at position 4(Fig. 2). The SS-8 isoform contains 11 residue repeats, similarto the winter flounder, with a high alanine content, which

accounts for the helicity. The helical wheel and helical netdiagrams highlight the amphiphilic character of SS-8 (Fig. 3).34

The winter flounder AFP is helical both in solution and inthe solid state. Circular dichroism studies of the winterflounder AFP revealed an a-helical conformation in aqueoussolutions at low temperature.35 The X-ray structure of thewinter flounder AFP in the solid state revealed it to be a singlehelix.29a,36 The helical periodicity places all hydrophilicresidues in the winter flounder AFP, such as Thr, on one faceof the helix, with a spacing between Thr residues around16.6 Å. Molecular modeling and docking studies showed thatthe face of the helix containing the alanine residues is involvedin ice binding, suggesting that hydrophobic interactions areinvolved.37 Sculpin AFP is more amphiphilic than winterflounder AFP as is evident from the higher hydrophobicmoment of the sculpin AFP compared to winter flounderAFP.38

II B. Type II AFPs

Type II AFPs are cysteine-rich non-helical proteins withoutrepetitive amino acid sequences and are large globularproteins with molecular weights that vary from 14 kDa to 24kDa. The species containing this class of protein include searavens, smelt, and American herring. Two main classes of typeII AFPs have been identified, based on their dependence onCa2+. The AFP from the sea raven is independent of Ca2+,whereas the AFPs from herring depend on Ca2+ for theiractivity.39

The sea raven AFP is one of the best-studied andcharacterized type II AFPs.40 It contains 18 different aminoacids, and a total of 163 amino acids with a molecular weightof 14–16 kDa. Biochemical characterization studies of the typeII AFPs are complicated by the presence of the five cysteineresidues. The structure of the sea raven AFP was solved usingNMR spectroscopy (Fig. 4a).41 The sea raven AFP includes 18%helices, 38% b-sheets, and 44% random coils. All 10 cysteineresidues present are involved in disulfide bond formation,which is vital for the protein’s stability and function.42

Antifreeze activity is reduced in the presence of dithiothreitol,highlighting the significance of disulfide linkages. The CDspectrum of sea raven AFP is complex, however, CD studiesindicated a low helix content, a high b-structure, and reverseturns. The negative bands at 300 nm and 292 nm in the CDspectrum of the sea raven AFP at 21 uC were predicted to arisefrom the presence of tryptophan residues, whereas a positiveband in the region between 250 nm and 285 nm was predictedto arise from tyrosyl and phenylalanyl residues.43 The region ofthe protein exposed to the aqueous environment contains thehydrophilic residues Thr, Asn, and Gln, each capable ofhydrogen bonding. The overall structural integrity of thisprotein is maintained by the disulfide bonds.

Type II AFPs from smelt and herring species are homo-logous to the C-type lectins, a type of carbohydrate-bindingprotein domain, where C-indicates the requirement of calciumfor binding (Fig. 4b).44 Herring AFP (hAFP) exists as monomerin solution as indicated by gel filtration and dynamic lightscattering studies. The bound Ca2+ is coordinated by Gln92,Asp94, Glu99, Asn113, Asp114 and a water molecule. Thecoordination geometry is pentagonal bipyramidal.

Fig. 3 (a) Helical net diagram of the SS-8 isoform with polar residues insidecircles,29 (b) helical wheel diagram representation from residues 8–45 of SS-8isoform,29 (c) a-helical structure of winter flounder type I AFP (PDB code wfb1).

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Substitution of Ca2+ with other metal ions resulted indecreased antifreeze properties and altered ice crystalmorphologies.45 Ca2+ coordination with the ice crystal latticeis believed to be involved in the ice growth inhibition. Type I,III and insect AFPs have larger ice-binding surfaces than hAFP.Crystal structure and site directed mutagenesis studiesrevealed the ice-binding site as Thr96, Thr98 and Ca2+

coordinating residues Asp94 and Glu99.

II C. Type III AFPs

Type III AFPs are globular proteins with a molecular weight of6–7 kDa that are devoid of any of the repetitive sequencesfound in type I AFPs. Type III AFPs are also devoid of cysteine,which is present in type II AFPs. Species containing type IIIAFPs include ocean pout, Antarctic eel pout, Arctic eel pout,and wolffish. Type III AFPs were first discovered in ocean poutand were found to be a mixture of twelve isoforms, whichcould be grouped into two distinct groups; quaternaryaminoethyl (QAE) and sulfopropyl (SP), depending on theirbinding affinity towards certain ion exchange resins.46 The SPand QAE isoforms are 55% identical. Among the twelveisoforms, eleven isoforms fell into the SP group and one fellinto the QAE group. The QAE group binds to QAE-Sephadex(QAE-1), and the SP group binds to SP-Sephadex (SP-1 to SP-4).The reverse phase HPLC of the SP-1 gives three peaks; HPLC 4,HPLC 5, and HPLC 6; SP-2 gives HPLC 1, HPLC 2, HPLC 3, andHPLC 11; SP-3 gives HPLC 8, HPLC 9, and HPLC 10; SP-4 givesHPLC 7, and the QAE-1 gives HPLC 12 (Fig. 5).

The secondary structure of the ocean pout AFP (QAEisoform) consists of eight b-strands arranged in three sheets.The NMR and X-ray crystal structure of type III AFP revealed acompact structure (Fig. 6a).47,48 The unique b-structure andmain-chain hydrogen bonds impart a rigid globular shape tothis protein. The protein buries a high fraction of itshydrophobic residues inside its core upon folding. Thehydrophilic areas between the b-strands were found to bindto the ice surface. The ice binding surface of the ocean pouttype III AFP was identified as the flat amphipathic region ofthe protein. Several hydrophilic residues, Gln9, Asn14, Thr15,Thr18 and Gln44 have been implicated in binding to ice. Theseresidues are flanked by the hydrophobic residues Leu, Ile, andVal, on the periphery and are also involved in ice-binding.49

These hydrophobic residues make favorable van der Waalscontacts with the ice surface due to shape complementarity

between the flat protein surface and the flat ice lattice.50 Theflatness of the ice binding sites is generally crucial to the icebinding mechanism. Interestingly, theoretical studies indicatethat the ice binding surface of less active SP isoform is flattestcompared to the more active QAE-isoform.51

The AFP from Antarctic eel pout comprises two type III AFPsjoined by a nine amino acid linker peptide (Asp-Gly-Thr-Thr-Ser-Pro-Gly-Leu-Lys) (Fig. 6b).52 This AFP, containing twosimilar AFP domains (dimeric), was reported to enhance theice growth inhibition activity compared to the correspondingmonomer. The finding was also supported by Langmuiradsorption isotherm models.52c This AFP assumes a compactglobular shape with two motifs arranged in pseudo-dyadsymmetry. Each motif contains four short b-strands, 310

helices, and an internal cavity.53 The recombinant dimer, inwhich each unit was connected by a peptide linker, displayedenhanced activity and this is attributed to the larger area of theice binding surface in contact with the ice.54

II D. Type IV AFPs

Type IV AFP was isolated from the longhorn sculpin from thecoastal waters of Massachusetts and New Hamsphire.55 This

Fig. 5 Classification of Type III AFP from ocean pout into twelve isoforms withion exchange chromatographic profiles on SP-Sephadex column and QAE-Sephadex column.46

Fig. 6 (a) X-ray crystal structure of ocean pout Type III AFP (PDB Code 1MSI).48

(b) Solution structure of dimer antifreeze protein RD3 (PDB Code 1c8a).52b

Fig. 4 (a) Solution structure of calcium independent type II AFP from sea raven,(PDB Code 2afp);41b (b) X-ray crystal structure of hAFP, the solid sphererepresents Ca2+ (PDB Code 2PY2).45b

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AFP, LS-12, is glutamate- and glutamine-rich (containing 17%of these amino acids) with a molecular weight of 12.3 kDa.Sequence analysis of the protein revealed that it contained 108amino acids and was blocked at the N-terminus by apyroglutamyl group. The sequence was confirmed by cDNAcloning and sequencing using liver RNA as the template.56

These studies showed sequence similarities to certain plasmaapolipoproteins, which assume helical bundles. CD studies ofthe type IV AFP indicated a high helical content (60% at 1 uC),and the protein folded as a four-helix bundle.57 The fouramphipathic helices were antiparallel and were oriented insuch a way that the hydrophobic surface formed the core of thebundle and the hydrophilic surfaces were directed outwardtowards the solvent (Fig. 7).58

A report by Hew and coworkers indicated that the longhornsculpin also produces a type I isoform AFP in its skin.59 Thus,the longhorn sculpin contains two types of AFP, type I and typeIV.

III. Antifreeze glycoproteins (AFGPs)

AFGPs refer to a group of eight structurally related glycopro-teins present in the blood serum of Antarctic notothenioidsand northern cod.60 Each AFGP contains various numbers ofrepeating units of (Ala-Ala-Thr)n, where n varies from 4 to 50,and a disaccharide b-D-galactosyl(1A3)-a-N-acetyl-D-galactosa-mine is attached as a glycoside to each of the hydroxyl oxygenmoieties of the Thr residues (Fig. 8).61

The electrophoretic properties of the AFGPs were used toclassify AFGP1–8.62 The AFGPs vary widely in molecularweights, from 2.6 kDa to 34 kDa (Table 2). AFGP1 is thelargest glycoprotein (34 kDa) and AFGP8 is the smallest (2.6kDa). AFGPs exist as a mixture of compounds rather than as asingle compound. For example, AFGP, isolated from rock cod,was found to have two fractions; a 6026–9784 Da fractioncharacterized by 14 different isoforms, and a 3865 Da fractioncontaining a single sequence.

Antarctic fishes contain y25 mg mL21 of AFGP, 25% ofwhich are AFGP1–5 isoforms, and 75% of which are AFGP6–8.The smaller AFGP6–8 from notothenioid differed to a smallextent with respect to their amino acid composition, where thefirst alanine in some of the repeats was replaced by a proline(Fig. 9a).63 In the AFGPs isolated from Arctic and NorthAtlantic cod, threonine is occasionally replaced by arginine;hence, the disaccharide attached to the hydroxyl group of

threonine in other AFGPs is absent in this AFGP at thisposition (Fig. 9b). The number and position of the proline andarginine residues in the smaller AFGPs are not generallyknown; hence, these AFGPs are represented as AFGP-ProX orAFGP-ArgX (X = 6–8).

Conformational studies of AFGPs were done using CD,NMR spectroscopy, and light scattering technique. Despite thedetailed spectroscopic studies, no 3D structure of any AFGPhas been published. This is primarily due to the existence of alarge number of conformers in solution. Based on the NMRand computational studies, it was concluded that AFGPs existas left-handed extended three-fold helices in solution. VacuumCD data also support the 3-fold left-handed helical conforma-tion.64 This conformation positions the disaccharides on oneside of the molecule and the relatively hydrophobic alanineresidues on the opposite side. The NMR data indicated thatthe disaccharide groups are tucked against the backbone ofthe polypeptide to form a stable hydrophilic surface capable ofinteracting with ice.65 13C NMR spectroscopic studies indi-cated that AFGP exists in flexible random coil conformation.66

The quasi-elastic light scattering (QELS) technique has shownthat conformations of AFGPs exist as extended coil struc-tures.67 In the presence of ice, AFGP2–5 and AFGP8 mostly

Fig. 7 Helical bundle structure of type IV AFP.58

Fig. 8 Various representations of AFGP (a) one letter code (b) three letters code(c) chemical structure.

Table 2 Distribution of molecular weights in AFGP1–862

AFGPs Approximate Mol. wt. Major Amino acids

AFGP1 34 kDa Ala, ThrAFGP2 29 kDa Ala, ThrAFGP3 21.5 kDa Ala, ThrAFGP4 17 kDa Ala, ThrAFGP5 10.5 kDa Ala, ThrAFGP6 7.9 kDa Ala, Thr, Pro or ArgAFGP7 3.8 kDa Ala, Thr, Pro or ArgAFGP8 2.6 kDa Ala, Thr, Pro or Arg

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display b-turn conformations. This conformational transitionis due to the high level of flexibility of AFGPs.

Conformations of the AF(G)Ps affect the TH values and theantifreeze activity. The TH of AFGPs is directly proportional toconcentration and chain length, with the larger glycoproteinsAFGP1–5 yielding higher TH values than the smaller AFGP6–8.68 AFGP8 was found to form a dimer in aqueous solutions atconcentrations exceeding 20 mM. The CD spectra of AFGP8indicated a random coil at low concentration, but a-helix andb-sheet structures were detected in a more concentratedsolution. Aggregated AFGP8 displayed a higher TH than asingle AFGP8 molecule, apparently due to cooperativeeffects.69

IV. Hyperactive AFPs

IV A. Hyperactive AFPs in insects

Terrestrial insects can survive under much colder conditions(230 uC) than polar fishes (21.9 uC).70 The two structurallynon-homologous AFPs in insects are beetle AFP and moth AFP.Tanebrio molitor (TmAFP) and Dendroides canadensis (DAFP),are the two beetle AFPs, which have been well characterizedand stuided in the last decade. Similarly, the well character-ized moth AFP is the spruce budworm (sbwAFP).71a–c TheTmAFP and sbwAFP contain b-helices and disulfide bondingpatterns.71d,e A comparison of the TH induced by fish AFPswith insect AFPs revealed that insect AFPs display a 10-foldhigher activity at one-tenth the AFP concentration of the fishAFPs and are hence referred to as the hyperactive AFPs. Thehyperactivity may be partly attributed to more effectivecoverage of the ice by insect AFPs permitting the recognitionof multiple ice planes.71f

DAFP has a molecular weight of 8.7–12.5 kDa and includesthirteen AFPs. Among these, DAFP-1, with 83 amino acids, andDAFP-2, with 84 amino acids, have been extensively studied.These AFPs contain seven repeating units, and each unit

contains 12 or 13 amino acids (-Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X13-).72 X3 and X11 are charged residues, X5(Thr or Ser), X6 (Asn or Asp), X9 (Asn or Lys), and X13 (Ala).The sixteen Cys residues present in this AFP are connected byeight disulfide bonds and account for 19% of the protein. Theamino terminal end contains pyroglutamate (pQ), and thecarboxyl end contains Pro. An additional Cys is present inDAFP-1 at position 10 in the first repeat and at position 4 inthe second repeat. The disulfide linkage in the first repeatforms between Cys-1 and Cys-10, whereas Cys-7 of this repeatis linked to Cys-4 of the second repeat. In other repeats, Cys-1typically connects to Cys-7 to form the disulfide bond.

The TmAFP has several isoforms. This AFP consists ofrepetitive patterns of the 12- or 13-reisdue repeats (-Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X13) that give rise to aright-handed b-helical structure.71d This 32 Å long b-helix ofTmAFP contains eight disulfide-linked twelve-amino acidrepeats. This AFP contains a regular array of Thr-Cys-Thr(TCT) motifs on one side. The crystal structure of the TmAFPindicates the presence of seven coils, of which six coils containsix b-strands and the seventh coil contains 14 residues and nob-strand. The average distance between hydroxyls within theTCT motif is 7.44 Å, whereas between equivalent hydroxyls ofTCT motifs in the adjacent loops is 4.64 Å (Fig. 10). The TCTmotifs form flat b-sheets and were found to be the ice bindingsurfaces.

The Thr hydroxyl groups align with the structures of boththe basal and primary prism planes of an ice crystal.73 Thehyperactive TmAFP produces ice crystals having the shape of alemon. A confocal microscope was used to image ice crystals inthe presence of GFP-conjugated TmAFP. The microscopicstudies with GFP-TmAFP demonstrated that TmAFP binds tobasal planes.74

The sbwAFP has a molecular weight of 9 kDa and containsrepeating patterns of 15-residue repeats. The repeat structureis less prominent, and only four disulfide bonds are present(Fig. 11). NMR studies revealed the presence of a b-helix with atriangular cross-section. The regular array of Thr-X-Thr motifs

Fig. 9 Structural representations of (a) AFGP-Pro and (b) AFGP-Arg.18g,63

Fig. 10 (a) Ribbon representation of the TmAFP b-helix (side view) with sixb-strands shown in green and disulfide bonds shown in yellow and (b) end-onview of the TmAFP b-helix (adapted with permission from ref. 71d copyright2000 Macmillan Magazines Ltd.).

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formed by the arrangement of the Thr residues (X is aninward-pointing amino acid) is the ice binding surface andmatches both the prism and basal planes hence the sbwAFPbinds to both prism and basal planes.71e The TCT motifs alignto form a flat b-sheet along one side of the molecule. The Thrresidues project outwards in two precisely aligned parallelarrays. The distance between Thr in neighbouring TCT motifsis 4.5 Å, whereas the distance between Thr in the TCT motif is7.35 Å. The b-helix is stabilized by a network of hydrogenbonds parallel to the helix axis linking the peptide CO and NHgroups of adjacent turns. The TH activity of sbwAFP is 3–4times that of the fish AFPs, and is 10–100 times more effectivethan the fish AFPs at micromolar concentrations.

Two AFP isoforms were isolated from the snow flea, thelonger one with a molecular weight of 15.7 kDa and the shorterone of 6.5 kDa. They have similar amino acid compositions.The short isoform of sfAFP contains 81 residues with four Cysresidues at positions 1, 13, 28 and 43 forming two disulfidebonds, 45.7% glycine and 13.6% alanine. Molecular modelingstudies indicate that the protein is organized into a bundle ofsix short polyproline type II (PPII) helices.75a The X-ray crystalstructure of sfAFP was determined by using racemic proteincrystallization, and was found to be made up of antiparallelleft-handed PPII helices.75b

Recently, the longhorn beetle Rhagium inquisitor is known toexpress AFPs in its body fluids. This AFP (RiAFP) has higherantifreeze activity compared to other known AFPs. RiAFPcontains 5 internal repeats of 7 residue pattern TXTXTXT,where X is small residue Ala or Thr.76a Based on the structureprediction algorithms, the TXTXTXT motif adopts a b-strandconformation. RiAFP contains a single disulfide bond. TheX-ray crystal structure of 13 kDa RiAFP reveals a b-solenoidfold.76b

IV B. Type I -hyperactive winter flounder AFP

Winter flounder was found to contain 10–15 mg mL21 of type IAFP during winter. This AFP accounted for only a 1.5 uC

decrease in the freezing point (0.8 uC depression contributedfrom the colligative effects of the solutes in the blood and 0.7uC depression from the non-colligative effect due to TH); butcould not account for the remaining 0.4 uC depression in theoverall 1.9 uC depression in the freezing point. Four decadesafter the discovery of winter flounder type I AFP, a re-evaluation of the TH activity led to the discovery of a newtype I AFP.77 The highly active AFP yields a large TH, leading tothe name ‘‘hyperactive’’ AFP. At room temperature, this AFPloses the majority of its TH activity.

The fractionation of winter flounder plasma by gel permea-tion chromatography on Sephadex G-75 reveals the presence ofseveral previously unidentified components. The highermolecular weight fraction elutes first, is present in lowquantities, and corresponds to the new hyperactive type IAFP. The lower molecular weight fractions elute later, displaytwo overlapping peaks, and are present in larger quantities.Reverse phase HPLC analysis of the overlapping peaks revealsthe presence of the HPLC-6 and HPLC-8 isoforms of type I AFP.The fraction containing the larger protein yields a high TH andaccounts for two-thirds of the total TH. The remaining one-third of the TH arises from the contributions of other smallerproteins. The large hyperactive AFP contains 195 amino acidswith a 60% alanine content, it has a molecular weigt of 16.7kDa (compared to 3.3–4.5 kDa for type I AFP), and it is moreactive than the type I AFP (HPLC-6 isoform).78 Circulardichroism studies reveal a dominant a-helical secondarystructure and it exists as a dimer in solution with a molecularweight of 32 kDa.

The TH activity of the hyperactive type I AFP in winterflounder is comparable to the activities of the TmAFP and thesbwAFP. These two hyperactive AFPs from insects predomi-nantly adopt a b-helical structure, in contrast with thehyperactive AFP isolated from winter flounder, which is ahighly elongated alanine-rich a-helical dimer.71d,e,78

IV C. Hyperactive bacterial AFPs

The presence of antifreeze protein in bacteria was firstdemonstrated by Duman and Olsen in 1993.15a The Antarcticbacterium Marinomonas primoryensis produces aCa2+dependent AFP; MpAFP, which has a molecular weightof 1.5 MDa.79 This protein contains five distinct regions(Fig. 12). The antifreeze activity of MpAFP is due to region IV(RIV). It contains two repetitive sequences that divide theprotein into five regions (I to V). Region II of this proteinaccounts for 90% of the total protein mass, containing 120tandem repeats of a 104 amino acid sequence.79b The RIV ofthis protein contains 13 tandem repeats of 19 amino acids(Fig. 12a). The consensus sequence is given as X1-G2-T3-G4-N5-D6-X7-U8-X9-U10-G11-G12-X13-U14-X15-G16-X17-U18-X19,where X is any amino acid (preferably hydrophilic) and U isany hydrophobic residue. The X-ray crystal structure ofrecombinant MpAFP_RIV shows a right-handed b-helicalstructure with Ca2+ bound on one side of the helix andhydrophobic core on the opposite side. The coil of the b-helixcontains one 6-residue Ca2+-binding turn and three shortb-strands separated by Gly-rich turns (Fig. 12b). The D6 of eachloop locks the Ca2+ ions into place and bridges each Ca2+

binding site. Thirteen Ca2+ ions align down one side of the

Fig. 11 (a) Ribbon representation of sbwAFP with b-sheets shown in bluearrows, disulfide bonds shown in green, Thr-rich face shown in red and (b)representation of sbwAFP showing the distribution of Thr residues (adaptedwith permission from ref. 71e copyright 2000 Macmillan Magazines Ltd.).

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structure and each one is heptacoordinated between succes-sive XGTGND turns (Fig. 12c)

The X-ray crystal structure of MpAFP reveals that the peptidebackbone makes hydrogen bonds with water molecules; theextensive array of ice-like surface water matches to the primaryprism and basal planes of ice. MpAFP_RIV contains a long andflat ice-binding site. The hydrophobic ice-binding site ofMpAFP arranges water molecules into an ice-like lattice, andthese ordered water molecules facilitate the binding of AFP tothe ice surface. This mode of binding is termed an ‘‘anchoredclathrate’’ mechanism.

V. Plant AFPs

AFPs are present in many over-wintering plants. The TH valuesexhibited by these proteins are generally low (about 0.2 uC to0.6 uC). These AFPs are therefore known to protect the plant byinhibiting ice recrystallization. Plant AFPs have multiplehydrophilic ice binding domains and thus can bind more icesurfaces. The AFP from winter rye (Secale cereale) is homo-logous to pathogenesis-related (PR) proteins, whereas the AFP(DcAFP) from carrot (Daucus carota) is homologous topolygalacturonase inhibitor protein.16 The DcAFP is found tobe N-glycosylated and has a leucine-rich structure.16b

The X-ray crystal structure of the AFP from ryegrass Loliumperenne (LpAFP) was reported recently.80 The 118 residueprotein folds as a left-handed b-helix with a length of 33 Å andwidth of 20 Å. The b-helical coil is composed of two seven-residue tandem repeats (XXNXVXG), where X is a residue witha polar side chain.

VI. Synthesis of native AF(G)Ps and theiranalogs

The chemical synthesis of natural AF(G)Ps or analogs withsimilar activities provides a viable route to the production ofsufficient quantities of pure AF(G)Ps. The development of

synthetic molecules that can mimic the structures andfunctions of natural AF(G)Ps is a challenging area of research.Such molecules can replace the natural AF(G)Ps in areas whereAF(G)Ps find applications. Moreover, these molecules canserve as good models for understanding the mechanism ofantifreeze action.

A 43-residue polypeptide 1 (Fig. 13) with mostly Ala and Lysresidues was designed and synthesized. This de novo design ofan AFP was based on the shorthorn sculpin and winterflounder AFPs. The design was based on the fact that alaninewould facilitate an a-helical structure, whereas lysine residuesfacilitate water solubility for binding to ice.81a This peptideexhibited the characteristic antifreeze property. Laursen et al.synthesized peptides 2 and 3 (Fig. 13) with regularly spaced Lysresidues.81b These peptides exhibited antifreeze activity.Interestingly, their sequences are very much different fromall known natural AFPs. Peptides with irregular spacing of Lysshowed no antifreeze activity.

Wang et al. reported a series of peptoids 4–6 (Fig. 13) withantifreeze activity.82 Studies showed that 5 inhibited the icegrowth more effectively than compounds 4 and 6, indicatingthe importance of hydrogen bonding and backbone structure.

The glycine rich snow flea AFP (sfAFP) is not available inlarge quantities and this hampered detailed mechanisticinvestigations. Kent and coworkers synthesized D-sfAFP (7),the mirror image form of sfAFP by employing sequential nativechemical ligations.83 The sequential ligation was done withN-terminal Cys incorporated as 1,3-thiazolidine-4-R-carboxylicacid (Thz) and thioester (COSR) at the C-terminal (Scheme 1).The enantiomers showed identical antifreeze properties.

Although the synthesis of AFGPs with relatively largemolecular weights and high sugar residue densities hasproven to be difficult, several groups have tackled thischallenge over the last twenty years and have worked outvarious solution phase and solid phase synthetic approachesto produce natural and synthetic AFGPs, as described in arecent comprehensive summary.84 Nishimura et al. devised asynthetic route to natural AFGPs via the solution phasepolymerization of an unprotected glycopeptide macromono-mer in the presence of diphenylphosphorylazide (DPPA)promoter.85 In subsequent years, the amino acid sequence of

Fig. 12 (a) Division of MpAFP into five distinct regions (I–V),79(b) consensussequence of MpAFP_RIV79 and (c) X-ray crystal structure of MpAFP_RIV, solidspheres indicate Ca2+ (PDB Code 3P4G).

Fig. 13 Various peptides and peptoids as synthetic mimics of AF(G)Ps81,82

Amino acid residues in the rectangles are based on winter flounder AFP.

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the repeat was changed from AAT to ATA under the notion thatbetter results could be achieved by reducing the sterichindrance.86 As the AAT repeats were changed to ATA, theTH increased, demonstrating the superiority of this design.Better results were also obtained by using other polymeriza-tion promoters; 1-isobutoxycarbonyl-2-isobutoxy-1,2-dihydro-quinoline (IIDQ) and 4-(4-6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM).87

An alternate synthetic approach using soild phase synthesiswas employed to prepare the natural AFGPs.88 The AFGPanalog 8 was synthesized using solid phase synthesis(Scheme 2). Fmoc-protecting group was used for N-terminalprotection. This strategy is effective in generating oligomerswith defined length and sequence variation compared to thesolution phase method. The loading of the first amino acidwas achieved by dicyclohexylcarbodiimide (DCC). Furthercoupling reactions were carried out using benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP). N-terminal Fmoc group was deprotected by 20%

piperidine. At the end of the synthesis, the cleavage from theresin was carried out using 95% TFA.

Nishimura et al. designed and synthesized a number ofAFGP analogs by using established synthetic methods.Antifreeze activity of these compounds was evaluated by usinga Clifton nanoliter osmometer and also by analyzing the icecrystal morphology. The structure–property relationships ofthe AFGPs were studied by synthesizing a variety ofcompounds in which the sugar units were substituted withgalactose and the stereochemistry of the glycosidic linkage waschanged (Table 3). A two-unit length (ATA)n repeat, (n = 2) wasfound to be sufficient to show TH activity, and the THincreased significantly with increasing chain length, up to n =5. However, no further increase in TH was observed among thesynthetic AFGPs from n = 5 to 7. This result did not agree withthe results obtained with the naturally occurring AFGPs. TheAFGP with n = 50 showed twice the TH as was obtained for n =5.89

A synthetic AFGP with a Ala-Ser-Ala repeat (Entry 1, Table 3)was synthesized to examine the importance of the c-methylgroups on the Thr residue. Although the ice crystal morphol-ogy changed to hexagonal, no TH activity was observed,suggesting that the c-methyl group of the Thr residue wasessential for TH activity.90 When the intra glycosidic linkagewas changed from 1–3 position to 1–4, (Entry 2, Table 3), THactivity remains unchanged. Feeney et al. observed that thecarbohydrate moiety was essential for TH activity, as evidencedby the complete loss of TH in the absence of the carbohydratemoiety (Entry 3, Table 3).63 The b-O-linked glycoprotein (Entry4, Table 3) showed no TH activity; hence the a-glycosidiclinkage between GalNAc and Thr is an essential structuralelement.90 Removal of the b-galactosyl unit from the dis-accharide retains TH (Entry 5, Table 3), indicating that thegalactose unit is not necessary for TH activity. The NHAc groupat the C2 position of the reducing end sugar residue isrequired for the antifreeze activity, as indicated in (Entry 6,Table 3). Overall, the results conclude that the antifreezeactivity of the AFGPs depends on the presence of an orderedsecondary structure such as PPII helix, a-configuration of theO-glycosidic linkage, c-methyl group of threonine and thepresence of N-acetyl group at C-2 position of the reducinghexosamine.

Nishimura et al. synthesized cyclic AFGPs 9 and 10containing a single peptide repeat and a disaccharide withdifferent numbers of repeats compared to the natural AFGPs(Fig. 14).91 Interestingly, despite the conformational differ-ences relative to the natural AFGPs, these cyclic analogsshowed ice shaping properties and TH activity. The TH,however, was independent of the molecular weight and thesmaller analog showed better activity than the larger analog.The retention of the TH activity in the cyclic AFGPs indicatesthat the C- and N-termini are not essential for hysteresisactivity.

Ben and coworkers synthesized C-linked analogs of AFGPstowards the generation of stable AFGP compounds, sinceO-linked analogs can undergo hydrolysis under acidic or basic

Scheme 1 Synthetic strategy employed for the synthesis of D-sfAFP 7.83

Scheme 2 Solid phase synthesis of 8.88

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conditions. Various modifications were applied to the naturalAFGPs to obtain the C-AFGPs; the (AAT) repeat of the naturalAFGPs was replaced by (Gly-Gly-Lys) in the C-AFGPs, and amonosaccharide was attached to the Lys in place of a

disaccharide attached to the Thr (11a–c, Fig. 15).92 Second-generation C-AFGP analogs (12a–c, Fig. 15) were designed toprobe the effects of the distance between the monosaccharidesand the peptide units on the antifreeze activity.92,93

Table 3 Different synthetic analogs of AFGPs and their antifreeze properties

Entry No. R1 R2 Ice crystal morphology TH Inference Ref.

1. H Hexagonal No c-methyl of Thr is essential 90

2. CH3 Hexagonal bipyramidal Yes Intra glycosidic linkage in thedisaccharide does not affect TH activity

90

3. H CH3 No change No Carbohydrate is essential 63

4. CH3 Hexagonal No a-glycosidic linkage betweenGalNAc and Thr is necessary

90

5. CH3 Hexagonal bipyramidal Yes Galactose is not necessary 90

6. CH3 No change No NHAc is essential 86

Fig. 14 Cyclic antifreeze glycopeptides.91 Fig. 15 Various C-AFGP analogs.92,93

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The synthesis of the C-AFGP analog 11a using solid phasesynthetic approach has been described in Scheme 3.92a

Tripeptide was coupled to the carbohydrate derivative andfurther hydrogenolysis provided the acid. This compound wasassembled using solid phase synthesis to afford the AFGPmimic.

A triazole unit was incorporated as a structural mimetic ofthe amide bond in some of the C-AFGPs to probe theimportance of the amide bonds on antifreeze activity

(Fig. 16).94 Brimble and coworkers synthesized a series oftriazole based AFGPs based on solid phase peptide synthesis.95

Sewald and coworkers synthesized a series of triazole-contain-ing peptoids employing on-resin click reaction.96 Longer AFGPanalogs were synthesized by using a combination of naturalchemical ligation and click reaction.97

All the natural antifreeze active compounds are based onamino acids, but recently a non-protein TH factor containingb-mannopyranosyl-(1–4)b-xylopyranose backbone and a fattyacid was isolated from Alaskan beetle Upis ceramboides.98 Itshowed TH of 3.7 uC (5 mg mL21) and strong ice crystalmorphology change.

Interestingly, an organometallic compound such as zirco-nium acetate, was recently found to display antifreezeactivity.99 This finding suggests several new directions in thesearch for potential antifreeze agents. The discovery andinvention of new antifreeze compounds may shed light on themechanism of action underlying the antifreeze phenomenonas well as providing commercially relevant antifreeze agents.

VII. Synthetic polymers

The difficulties faced in the synthesis and purification ofAF(G)Ps coupled with their lower stability resulted in theirlimited commercial applications. However, the structural andfunctional tunability of the synthetic polymers over a wider

Scheme 3 Solid phase synthesis of C-AFGP analog 11a.92a

Fig. 16 Various synthetic triazole-based C-AFGP analogs 13 synthesized by Ben and coworkers;94 triazole-based AFGP analogs 14 synthesized by Brimble andcoworkers;95 triazole-based peptoids 15–17 synthesized by Sewald and coworkers.96

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range makes them an attractive candidate to mimic theproperties of AF(G)Ps.100,101 Therefore, various approaches tomimic the properties of AF(G)Ps using non-peptidic syntheticpolymers were undertaken. Table 4 shows some of thesynthetic polymers showing antifreeze properties.

Poly(vinyl alcohol) and other synthetic polyols showantifreeze activity.102a The interatomic distances between theoxygen atoms of the hydroxyl groups in the polyols are acritical factor for antifreeze acivity.102b The TH activity ofpoly(vinyl alcohol) is concentration dependent. It has alsobeen reported that poly(vinyl alcohol) adopts a multiple-stranded helical structure due to hydrogen bonding andmaintains a rigid structure required for exhibiting THactivity.102c However, poly(acrylic acid), poly(ethylene glycol)and poly(vinyl pyrrolidone) do not show any TH activity.Interestingly, ammonium polyacrylate showed TH beha-vior.103a,b Hence, the ammonium ion is predicted to play arole in the antifreeze activity.103b Polymers containing poly-ethyleneimine (PEI) and poly(ethylene glycol) (PEG) incorpor-ating poly(glycidol) (P(Gly)) groups were found to show THactivity.103a,103c PEI23-Gly and PEG113-b-PEI23-Gly have beenreported to show a TH of 0.69 uC (1 mg mL21) and 0.83 uC (1mg mL21), respectively.

VIII. Mechanism of action underlying theantifreeze properties

The freezing point is the temperature at which the solid andliquid phases of a substance are in equilibrium at atmosphericpressure. Freezing point depression typically arises from thecolligative properties of a solute. When a solute is dissolved ina solvent, the freezing point of the solution is loweredaccording to the equation,104

DT = iKfm, where

DT = Change in temperature

i = van’t Hoff factor

m = molality

Kf = molal freezing point constant

Freezing in a solution requires that the molecules cometogether and form a cluster that further grows into a solid. Thepresence of a solute in the solvent disrupts the packing of thesolvent molecules. This disruption is counterbalanced byenthalpic considerations at lower temperatures, resulting in alower freezing point relative to the freezing point in theabsence of the solute.

Blood acts as a buffer solution and is expected to displayfreezing point depression based on the colligative properties ofa variety of solutes therein. The large freezing point depressionobserved in the blood of polar fishes exceeds the expectedvalue predicted by the colligative properties of blood alone.Therefore, the freezing point depression of the blood of polarfish must arise from the combined effects of the colligativeand non-colligative properties (Fig. 17).22 The colligativecontributions of the solutes in the plasma account only for adepression between 0 and 20.7 uC. The remaining freezingpoint depression is achieved by AF(G)Ps. Investigations byseveral research groups have clearly established that theantifreeze activity of these proteins functions in a non-colligative way.12,105

A number of theoretical investigations have been under-taken to understand TH activity of AFPs in considerabledetail.106 Based on many studies, it is argued that AFPirreversibly binds on to the ice surface. Irreversible binding

Table 4 Different synthetic polymers and their antifreeze activities

Polymers TH/uC Ice crystal morphology change Refs.

Poly(vinyl alcohol) 0.037 (50 mg mL21) weak 102Ammonium polyacrylate 0.043 (300 mg mL21) weak 103(PEI-Gly)23 0.69 (1 mg mL21) weak 103PEG113-block-(PEI-Gly)23 0.83 (1 mg mL21) strong 103

Fig. 17 Illustration of freezing point depression observed in polar fishes. (a) Thedepression in freezing point is a result of the combination of colligative andnon-colligative effects,22 the colligative depression is from 0 uC to 20.7 uC. (b)Representation of thermal hysteresis gap; within the thermal hysteresis gap, icewill not grow.105c

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is very unusual in a typical binding scenario. It is believed thatthe antifreeze molecules with large surface area bind onto theice surface with a number of hydrogen bonding and hydro-phobic interactions. Breaking these large number of noncovalent contacts simultaneously is unlikely. Apart from that,the activation energy for this will be large and therefore thedesorption rate is considered to be zero.107

Adsorption–inhibition via adsorption to an ice crystalsurface is one of the most incredible and difficult molecularrecognition tasks achieved in biology. The receptor (AFP)–ligand (ice) recognition relies on the alignment between theice planes and the complementary protein surfaces. Thehexagonal form (Ih) is the most common form of ice andcontains two basal faces normal to the c-axis and six prismfaces parallel to the c-axis (Fig. 18). Ice growth predominantlyoccurs from the prism faces.

AFPs adsorb on to a particular set of ice planes and inhibitthe crystal growth normal to the bound surfaces. The crystalscontinue to grow on unprotected planes. The binding of AFPsto different crystallographic ice planes was determined bygrowing single ice crystal hemispheres. The protein bindingplanes of ice were visualized in the ice etching experiments.108

The etching patterns obtained on the surfaces of the icehemispheres suggested that different AFPs have differentspecificities for certain ice planes and directions. Type I AFPfrom winter flounder and Alaskan plaice AFPs adsorbed ontothe (2021) pyramidal plane of ice, whereas the shorthornsculpin AFP adsorbed onto the (2110) secondary prism plane(Fig. 18b–c)).105,108

Ice grown in the presence of fish AFPs (Type I, II or III)formed a hexagonal bipyramidal structure (Fig. 19).38 Icecrystal growth inhibition by the winter flounder AFPs occurredin a two-step process involving the hydrogen bonding of Thr,Asn, Asp side chains to ice and hydrophobic interactions.109

The underlying mechanism involving a combination ofhydrogen bonding and hydrophobic interactions betweenAF(G)Ps and ice has been debated for over 30 years. Initially,researchers proposed that the spacings between the hydroxylgroups of Thr (16.6 Å) in the winter flounder AFP matched theoxygen–oxygen spacing of 16.7 Å on the prism face of the icelattice, thereby favoring the binding of AFP to the pyramidalplane of ice and was further supported by computationalstudies.110 A zipper-like model involving hydrogen bonds

between the Thr residue and the oxygen atoms of the ice latticeis shown in Fig. 19b. Computational studies showed that thehydroxyl groups of the four Thr residues Thr-2, Thr-13, Thr-24,and Thr-35 in winter flounder AFP (the HPLC-6 isoform ofType I AFP) aligned with the helical axis and were equallyspaced, i.e., 16.1 Å, 16.0 Å, and 16.2 Å, respectively.110

More recently, it was postulated by several research groupsthat the Thr hydroxyl groups were not involved in ice binding;rather, the methyl groups were involved.111 To shed light onthe importance of the hydrophobic groups as well as thehydrogen bonding groups, several modification studies wereconducted. The Thr-13 and Thr-24 in winter flounder (HPLC-6isoform of Type I AFP) were changed to Ser and Val. The Sersubstitution resulted in a complete loss of antifreeze activity,whereas the Val substitution produced little loss in activity,suggesting that the methyl group, and not the hydroxyl group,in Thr was responsible for the antifreeze activity. The mutationof Ala-17 to Leu led to the complete loss of antifreeze activity;hence, it was predicted that the alanine-rich face, in additionto the methyl groups of threonine, contributed to the icebinding face.22b

Knight et al. looked at all possible interactions of AFGPwith ice in order to explain the irreversible binding.112 Theresults supported a model in which only two hydroxyl groupsof each disaccharide in AFGP take part in binding to ice. In theproposed model, the disaccharide hydroxyl becomes part ofthe ice lattice and each makes three hydrogen bonds withwater molecules (Fig. 20). For AFGP-8, eight hydroxyl groupsfrom four disaccharide units take part to make a total of 24hydrogen bonds. This supports the proposed irreversiblebinding of AFGP to ice.

Hysteresis behavior could be explained in terms of theadsorption of AFPs to an ice surface leading to the growth ofice in the regions between the adsorbed AFPs (Fig. 21). Duringcooling the ice front advances, and most solutes, including thevast majority of proteins, are excluded and pushed ahead of

Fig. 18 Representations of (a) different faces of the hexagonal polymorphicform of ice; (b) 2021 adsorption plane; (c) 2110 adsorption plane of ice.108

Fig. 19 (a) Cartoon representation of the mechanism of hexagonal bipyramidalice crystal formation as a result of AFP binding.38 (b) Zipper-like model showingthe hydrogen bonds between Thr residues.110.

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the expanding ice front. AF(G)Ps behave very differently. Theybind to the ice and restrict the growth of an ice front in theregion between adsorbed protein molecules. These regionsgrow with a surface curvature that is thermodynamicallyunfavorable. The curved ice surface incurs a higher surfacefree energy, making it thermodynamically unfavorable forwater molecules to add to the ice lattice. The thermodynamicpenalty of the increased curvature restricts the growth of theice front in regions between adsorbed AFP molecules. Thecurvature-induced effect is called the Kelvin effect(Fig. 21).18a,105,113

Two major models, the mattress button model and step-pinning model, are currently used for rationalizing theantifreeze activity of AF(G)Ps.18i,108,113 In the mattress buttonmodel, individual AF(G)Ps are considered as buttons that bindto the ice-surface, limiting the ice growth to spaces betweenadsorbed AF(G)Ps, resembling a mattress-like surface. In thesecond model, the step-pinning model, AF(G)Ps are believed tobe ‘‘pinned’’ to the surface of ice and thus inhibit the icegrowth. Ice growth inhibition occurs in both models throughthe Kelvin effect.

The nucleation–inhibition mechanism was found to oper-ate via AFP binding to the surfaces of ice nuclei and dust

particles.114 Freezing is a process in which ice crystallizes fromsupercooled water. Nucleation, followed by crystal growth, aresteps towards the formation of ice. Without the nucleationstep, ice will not form in supercooled water. Dust particles canpromote ice nucleation; therefore, to inhibit ice nucleation,AFPs should be able to disrupt the interactions between icenuclei and foreign particles. Under normal conditions, icenucleation by dust particles cannot be eliminated. Tominimize the effects of dust particles, the water used in theexperiments was filtered twice using 20 nm filters to removelarge particles. The freezing temperature was found todecrease progressively as the pore size of the filters decreasedfrom 200 nm to 100 nm and then to 20 nm. This observationimplied that dust particles influence ice crystallization andshould be considered in any study. The nucleation kinetics ofice may be altered by the binding of AFP molecules to thesurfaces of either dust particles or ice nuclei. AFP moleculesare similar to surfactant molecules and, therefore, can self-assemble on the surface of a water droplet while maintainingthe hydrophobic surface orientation away from the droplet.This conformation reduces surface tension and has beenconfirmed experimentally. Similarly, AFPs can self-assembleon the surfaces of dust particles.

The antifreeze activity of type I AFP composed entirely of D-amino acids showed identical biological activity to that of wildtype.115 Molecular recognition occurs between a chiral AFPand a non-chiral ice substrate; therefore, chirality does notplay a crucial role in binding, unlike many natural processesthat are strictly stereospecific.

IX. Applications

AF(G)Ps protect cells, tissues, and cell membranes fromhypothermic damage, and therefore have many potential usesin medicine and food industries.116 The difficulties associatedwith isolating natural AF(G)Ps in pure forms and largequantities have hampered the exploration of many of theirpotential applications. Therefore, the synthesis of novelmolecules with antifreeze properties is an alternate approachto overcome this problem. Engineering AF(G)Ps to gain higherantifreeze activity is another way that their potential applica-tions might be enhanced. Transgenic plants with AFPexpressed in their fruits were also explored.117 The introduc-tion of AF(G)Ps into the organism through genetic engineeringmethods is currently a topic of debate.

AF(G)Ps find applications in the cryopreservation ofbiological samples such as cells, tissues and embryos. Thecurrent duration for the storage of donor organs fortransplantation is limited. AFPs may enhance the storagetime by slowing cellular degradation. The major cause ofcellular damage in cryopreservation is the recrystallization ofice. Reducing the rate of ice crystal growth would be expectedto improve the preservation time. AF(G)Ps can protectmammalian oocytes and porcine oocytes during cryopreserva-tion.118

Fig. 20 (a) Hydrogen bond formation between disaccharide hydroxyl of AFGPand ice. (b) In this model, the hydroxyl groups of disaccharides are incorporatedinto the ice lattice. Three hydrogen bonds per one hydroxyl group areformed.112

Fig. 21 Schematic illustration of ice growth inhibition by AFPs (dark spheres).The curvature between the bound AFP molecules leads to ice growth inhibitionby the Kelvin effect.18a

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Cryosurgery is an emerging method for treating cancersand tumors. It is a surgical technique used to destroyundesirable tissues via freezing. This method relies onintracellular ice formation. The addition of AFPs can enhancethe cryoinjury in cancer cells through mechanical damage tocells due to the growth of spicular ice crystals and thus can beused as a therapy.119

One of the other attractive potential applications of AF(G)Psis in food storage. Freezing of food items such as strawberries,raspberries and tomatoes results in loss of quality due tocellular destruction caused by ice formation. The presence ofAFP can maintain the smaller ice crystals during freezing,which may protect cellular damage and thereby preserve thequality of frozen food.117 Meat can similarly be preservedwithout loss of quality. Unilever has used AFP commercially inice cream to preserve its texture.120

The properties of AFPs offer opportunities for variousnanotechnological applications. A coating based on AFPsshowed improved ice-repellent properties.121 Such coatingswill be useful in transporting vehicles such as ships,automobiles and aircrafts.

X. Conclusions

AFPs and AFGPs are nature’s molecules of choice forprotecting against cold climatic conditions. The presence ofthese proteins in fishes, insects, bacteria, and plants,particularly in a diverse array of structures, presents one ofnature’s architectural marvels. The molecular mechanism ofaction underlying the function of these proteins is not yet fullyunderstood. The incredible molecular recognition propertiesof AF(G)Ps with respect to binding to particular faces of ice isthe envy of chemists and biologists. Several experimental andtheoretical approaches have been applied towards elucidatingthe structure–function relationship. Bioinspired moleculardesign will hopefully provide new insights into the molecularrecognition and the TH properties. The synthesis of moleculesdesigned to optimize ice binding and TH is in its infancy andmay prove to be successful in the coming years. Applications ofantifreeze agents in cryosurgery and the food industry provideadditional motivation for chemists towards the design andsynthesis of artificial antifreeze agents.

Acknowledgements

We thank the Department of Science and Technology (DST),New Delhi for the financial assistance.

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