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Insect Biochemistry and Molecular Biology 41 (2011) 109e117

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Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier .com/locate/ ibmb

Structural characteristics of a novel antifreeze protein from thelonghorn beetle Rhagium inquisitor

E. Kristiansen a,b,*, H. Ramløv b, P. Højrup c, S.A. Pedersen a, L. Hagen d, K.E. Zachariassen a

aDepartment of Biology, Norwegian University of Science and Technology (NTNU), Trondheim, NorwaybDepartment of Science, Systems and Models, Roskilde University, Roskilde, DenmarkcDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, DenmarkdDepartment of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway

a r t i c l e i n f o

Article history:Received 29 March 2010Received in revised form5 November 2010Accepted 6 November 2010

Keywords:Antifreeze proteinAFPIce-binding motifSequenceInsectBeetleRhagium inquisitor

* Corresponding author. Department of Science, SyUniversity, Universitetsvej 1, P.O. Box 260, DK-400046743138; fax: þ45 46743011.

E-mail address: erlendk@ruc.dk (E. Kristiansen).

0965-1748/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.ibmb.2010.11.002

a b s t r a c t

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and areproduced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold envi-ronments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetleRhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primarystructure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity toevoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in severalaspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs,RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat patternis irregularly spaced apart throughout its sequence. The central part of these repeat segments ispreserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs ofseveral known insect AFPs.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Antifreeze proteins (AFPs) are defined by their capacity to causea separation of the melting- and growth-temperatures of ice,known as thermal hysteresis. These structures have evolved inde-pendently in a variety of organisms that may experience super-cooling of their body fluids, including many species of polar fish(Raymond and DeVries, 1977), spiders and insects (Zachariassenand Husby, 1982; Duman et al., 2004). The antifreeze activityreportedly varies from about 1 �C in blood of polar fish (Raymondand DeVries, 1977) to 5e7 �C in the hemolymph of many over-wintering insects (Duman et al., 2004).

The antifreeze activity of AFPs combined with their exclusiveoccurrence in cold-hardy organisms suggests that they protectagainst lethal freezing of the body fluids. Zachariassen and Husby(1982) found evidence indicating that AFPs from the cerambycidbeetle Rhagium inquisitor may cause an antifreeze activity thatcorrespond to the supercooling range of this species. Olsen and

stems and Models, RoskildeRoskilde, Denmark. Tel.: þ45

All rights reserved.

Duman (1997) and Duman (2002) showed that AFPs from Den-droides canadensis mask the effect of structures that triggerfreezing. Olsen et al. (1998) also showed that AFPs prevent externalice from inoculating body fluids through the body wall.

Despite the fact that many species of overwintering insects havebeen reported to express antifreeze activity (Duman et al., 2004)there is structural information on insect AFPs only from the twospecies of beetles Tenebrio molitor (Liou et al., 1999, 2000a,b) andD. canadensis (Andorfer and Duman, 2000), the lepidopteran genusChoristoneura (Doucet et al., 2000, 2002; Tyshenko et al., 2005) andthe snow flea Hypogastrura harveyi (Graham and Davies, 2005; Linet al., 2007; Pentelute et al., 2008). The primary structures of theAFPs from the tenebrionid beetle T. molitor and the pychroid beetleD. canadensis are quite similar and are organized as a continuousseries of 5e8 connected 12- or 13-mer repeat regions, bothwith theconsensus sequence TCTxSxxCxxAx (Li et al., 1998a,b; Liou et al.,1999, 2000a; Andorfer and Duman, 2000). Notably, every sixthresidue within the repeat sequence is a cysteine and the twocysteines within the repeat form a disulfide bridge. Those ofT. molitor have been shown to fold into a right-handed b-helixwhere every 12-mer repeat region forms one turn in the structure.Its disulfide bridges cross the helix, contributing to the tightness ofthe structure (Liou et al., 2000b). Several species of the Lepidop-teran genus Choristoneura express AFPs with primary sequences

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117110

that show no homology to the beetle AFPs, have no obvious repeatsegments and contain four disulfide bonds (Tyshenko et al., 2005).The AFPs of Choristoneura fumiferana fold into a b-helix structure,but this helix is left-handed rather than right-handed, and eachturn of the helix consists of 15 amino acid residues rather than the12-residue turn seen in the beetle AFPs (Graether et al., 2000).Recently, structural characteristics of an AFP from the snow flea,H. harveyi, revealed an amino acid sequence that consists ofa continuous series of the tripeptide G-x1-x2, where x1 is usually Gand x2 is A or V (Graham and Davies, 2005). The structure containstwo disulfide bridges and its proposed secondary structure showsno homology to those of the coleopterans or lepidopterans, beinga bundle of six short anti-parallel polyproline type II helices thatform two sheets of three parallel helices oriented anti-parallel toeach other (Lin et al., 2007; Pentelute et al., 2008).

Although their structures are diverse, all AFPs are thought toevoke the antifreeze effect in a similar manner, by becoming boundto the surface of the ice crystal. This explanation has been coinedthe adsorption-inhibition hypothesis (Raymond and DeVries,1977).According to this view, the exposed ice surface regions between theadsorbed AFPs grow out as convex interfaces as the temperature islowered. This convex growth pattern in turn elevates the ice vapourpressure of these growth regions, i.e. the Kelvin effect, (Thomson,1871), and growth therefore stops once their vapour pressure iselevated to that of the surrounding solution (Kristiansen andZachariassen, 2005). Consequently, iceewater vapour pressureequilibriumwill persist within a temperature interval. At some lowtemperature, however, the convex surface zones reach a maximumconvexity, ideally that of a half-sphere. Any further coolingwill onlyresult in the growth zone expanding beyond its base, causing theconvexity of the growth zones to decrease rather than increase andthe ice crystal will grow explosively. This temperature at which theantifreeze phenomenon is terminated is called the hysteresisfreezing point and the antifreeze activity is defined as the differ-ence between the equilibrium melting temperature of the icecrystal and the hysteresis freezing point. In this model, antifreezeactivity increases with the maximum obtainable convexity of thegrowing interfaces between the adsorbed AFPs, i.e. decreaseddiameter of the sphere. Consequently, antifreeze activity is thoughtto increase with decreased spacing between the adsorbed AFPs,consistent with the observations that the antifreeze activityincreases with increased concentrations of AFPs.

In support of the adsorption-inhibition hypothesis, AFPs containspecific sites at their surface which show a remarkable structuralmatch to that of ice (Liou et al., 2000b; Jia and Davies, 2002). Theputative ice-binding sites of the AFPs from both the beetlesT. molitor and D. canadensis and those of the lepidopteran genusChoristoneura are made up of a stack of the triplet threonine-x-threonine, known as the ‘TxT’-motif, which together forms a flatsurface area on one side of the protein (Liou et al., 2000b; Graetheret al., 2000). The hydroxyl groups of the twoT-residues within eachTxT-motif, together with tightly bound water molecules, areoriented in such a way that they mimic the ice structure. Theproposed ice-binding site of the snow flea AFP consists of one of thethree-helix-sheets that form a flat hydrophobic side (Lin et al.,2007).

The cerambycid beetle R. inquisitor is freeze avoiding and maysupercool to below �25 �C in the cold-hardy state (Zachariassenet al., 2008). It overwinters under the bark of dead stems andstumps of spruce and pine throughout Europe and even in theextremely cold parts of Siberia. It expresses AFPs both within itstissues and its hemolymph (Zachariassen and Husby, 1982;Kristiansen et al., 1999). This study reports on structural char-acteristics of the dominant AFP isoform found within itshemolymph.

2. Material and methods

2.1. Collection and handling

Cold-hardy larvae of the beetle R. inquisitor were collectedduring winter from their hibernation sites under the bark ofstumps and logs of spruce, Picea abies, in the vicinity of Trond-heim, Norway. The larvae were transported to the laboratory onice. Larvae to be sampled for hemolymph were kept in thelaboratory at 0 �C for up to one week prior to sampling, whereaslarvae used for RNA extraction were processed immediately uponreturn to the laboratory.

2.2. Hemolymph sampling and protein isolation

A small incision was made on the left side of the frontalprothorax region of the larvae with a pair of surgical scissors. Theattenuated end of a flame drawn capillary tube was then used tocollect the exuding hemolymph by means of the capillary force.Once filled with hemolymph the capillary tube was placed ina small hole in the lid of a 1.5 mL Eppendorf tube containingapproximately 200 ml of liquid paraffin oil. Brief centrifugation atlow speed left the hemolymph at the bottom of the Eppendorf tube,isolated from the air by the less dense paraffin oil. The Eppendorftube was kept on ice between centrifugations. The sampledhemolymph was stored at �40 �C awaiting further processing.

The dominant AFP isoform in the hemolymph of R. inquisitor,denoted RiAFPH4 and abbreviated here to RiAFP, was isolated byheat and acid extraction followed by cation ion exchange chro-matography (IEX) as previously described (Kristiansen et al., 2005).The fractions from the IEX procedure containing purified RiAFPwere freeze-dried and stored at �40 �C.

2.3. Determination of antifreeze activity

The concentration of RiAFP in a stock solution was determinedby amino acid analysis and diluted in 0.9% NaCl to the desiredconcentration. Small aliquots of the dilutions were kept at �35 �Cprior to use.

Using a Clifton nanoliter osmometer (Clifton Technical Physics,USA), approximately 10 nl droplets were submerged in liquidparaffin and frozen by rapid automatic cooling to �40 �C. Thefrozen droplets were then heated to a preset temperature some-what below the expected melting point of the sample. Whileobserving the sample through a microscope, the temperature wasslowly raised (w0.5 �C/min) until only a single small ice crystalremained, with an approximate diameter of 7 mm. The temperaturewas then kept unchanged for 1 min to allow the crystal to stabilize,followed by slow cooling (w1 �C/min) until rapid ice growth wasobserved. This temperature of ice growth was taken as thehysteresis freezing point and the difference between the stabili-zation temperature and the hysteresis freezing point was taken asthe antifreeze activity.

2.4. Determination of the primary sequence of RiAFP

Overall, the strategy to obtain the complete primary sequence ofRiAFP was as follows: First, obtain the amino acid sequence offragments of RiAFP by MS and Edman degradation followingenzymatic digest. Second, determine a partial cDNA sequencefollowing PCR by the use of degenerate primers constructed on thebasis of the obtained amino acid sequences from the initial peptidefragments. Third, use the partial cDNA sequence to construct gene-specific primers to determine the remaining parts of the DNAsequence by rapid amplification of cDNA ends (RACE).

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117 111

2.4.1. Amino acid sequence determination2.4.1.1. Reduction, alkylation and enzyme digestion. Protein aliquotsof approximately 50 mg RiAFP were redissolved in 40 ml 6 Mguanidine HCl and 40 ml 0.1 M DTT in TriseHCl pH 8.6 for 2 h at56 �C followed by the addition of 40 ml 55 mM iodoacetamide.Aliquots ofw50 mg RiAFP were incubated with 2 mg trypsin or Lys-Cat 37 �C overnight. Finally, 10 ml formic acid was added and thesample stored at �20 �C.

2.4.1.2. RP-LC of enzyme digests. The enzyme digest was separatedon an Ettan LC system (GE Healthcare) using a Jupiter 1 � 150 mmC18 RP-HPLC column (Phenomenex) thermostated at 45 �C. Elutionwas carried out using gradients of (A) 0.1% (v/v) TFA and (B) 90%acetonitrile, 0.08% TFA for 45 min. The UV-detector was set at 214,254 and 280 nm and fractions were collected manually. Eachcollected fraction was dried and diluted with 0.1% TFA in water toa final concentration of w1 pmol/ml.

2.4.1.3. Micro-purification columns. Reversed phase micro-purifi-cation columns were prepared from GELoader tips as described byGobom et al. (1999) using a suspension of Poros R2 resin inacetonitrile (PerSeptive Biosystems, USA). The columnwas washedonce with 10 ml of 0.1% TFA, 10 ml of acetonitrile and then equili-brated with 10 ml of 0.1% TFA prior to use.

About 5 ml of resuspended fractions from the RP-HPLC orenzyme digest was loaded onto the micro-purification column andwashed with 5e10 ml of 0.1% TFA. The adsorbed peptides could nowbe eluted for routine MS analysis of RP-HPLC fractions or be furthermodified by the Chemical Assisted Fragmentation (CAF) procedure(see below) for de novoMSmicrosequencingwhile still adsorbed onthe column. Elution from the column was in all cases done directlyonto the steel target for MALDIMS by a volume of 0.5e0.8 ml MALDImatrix (20 mg a-cyano-4-hydoxycinnanic acid/ml (70% acetonitrile,0.1% TFA)). Routine MS analysis of RP-HPLC fractions was doneusing an Applied Biosystems Voyager Elite operated in the positivereflector mode with 20 kV accelerating voltage.

2.4.1.4. Chemical modification for de novo MS microsequencing. CAF-modification was carried out using an Ettan CAF-MALDI SequencingKit (Amersham Biosciences). O-Methylisourea hydrogen sulfatesupplied with the kit was replaced by O-Methylisourea hemisulfate(Beardsley and Reilly, 2002).

2.4.1.5. Guanidination of lysine to homoarginine. Approximately 1/10of each resuspended RP-HPLC fraction was applied to the micro-purification column and washed with 5 ml 0.1% TFA. Then 5 ml lysinereagent solution (4.5 ml lysine reagent (10 mg o-methylisoureahemisulfate in 10.2 ml H2O), 16.5 ml lysine modifier buffer (7 MNH4OH), and 15 ml H2O) was added and the column heated to 65 �Cfor 10 min. The remaining lysine reagent solutionwas then removedand the column washed with 3 ml 0.1% TFA.

2.4.1.6. Sulfonation of N-terminus by CAF reagent. The column waswashed with 2 ml CAF buffer (0.25 M sodium bicarbonate, pH 9.4).Then 1.5 ml of CAF reagent solution was added (1 CAF vial added60 ml H2O), and the reactionwas allowed to proceed for 3min. A 3 mlstop solution (5% hydroxylamine diluted in H2O) was then added.After 1 min the stop solutionwas removed and the columnwashedwith 3 ml 0.1% TFA.

2.4.1.7. De novo microsequencing. Protein microsequencing of theindividual CAF-modified fragments was performed on a MALDIQuadruple Time-of-Flight (QTOF) mass spectrometer (Micromass,Manchester, UK). Argon was used as collision gas at an indicatedmanifold pressure of 3 � 10�5 mBar in the hexapole and the

collision energy adjusted to 70e100 eV. The resultant mass spectrawere generated by MassLynx 3.5.

2.4.1.8. Edman degradation. The native protein and fractions fromthe RP-HPLC following enzyme digestion were applied to a Bio-brene precycled glassfiber filter and sequenced by automatedEdman degradation using a pulsed phase sequencer (Applied bio-systems 477A). The resultant PTH derivatives were identified usingan on-line PTH analyzer (Applied Biosystems 120A).

2.4.2. DNA sequence determination2.4.2.1. Extraction of total RNA. To preserve mRNA, immediatelyupon return from the field to the laboratory 10 larvae werehomogenized in TRIzol reagent and the supernatant stored at�80 �C until further processing. Total RNAwas purified as describedby the manufacturer (Invitrogen Corporation, Carblad, CA, USA).Potentially contaminating DNA was removed by DNAase I treat-ment (DNA free kit, Ambion, Austin, Texas, USA). Total RNA yieldwas determined at 260 nm using a NanoDrop ND-1000 spectro-photometer (NanoDrop Technologies, Wilmington, USA).

2.4.2.2. Degenerate PCR. For amplification of the core cDNA frag-ment of RiAFP, the degenerate primers RiAFP_F1 (50-TGYMGIGCIGTIGGIGTIGAYGG-30) and RiAFP_R1 (50-GTYTGIGTYTGIGTIGGYTG-30)were designed based on the amino acid sequencesmade available bymeans of protein sequencing methods. Approximately 1 mg of totalRNA and 30 mmol of the two degenerate primers RiAFP_F1 andRiAFP_R1 were used in a 50 ml OneStep Reverse Transcriptase (RT)PCR reaction (Qiagen GmbH, Hilden, Germany). The temperatureprogram included a reverse transcription step at 50 �C (30 min),a heat activation and denaturation step at 95 �C (15 min), 40amplification cycles at 94 �C (30 s), 50 �C (1 min), 72 �C (1 min),followed by a final extension step at 72 �C (7 min). Inspection of thereaction using agarose electrophoresis and ethidium bromidestaining revealed a single strong bandwith an apparent size ofw350base pairs. The band was cut out from the gel and the PCR productpurified using the QIAquick Gel Extraction Kit (Qiagen). The purifiedPCR-product was ligated into the PCR�2.1 vector and used to trans-form chemically competent cells (INVaF0, Invitrogen). Positive cloneswere detected using blue/white screening, grown overnight andharvested for plasmid purification using a GenElute Plasmid Mini-prep kit (SigmaeAldrich Inc.). The purified plasmid was prepared forsequencing using the BigDye� Terminator v1.1 Cycle Sequencing Kit(Applied Biosystems, CA, USA) and M13 reverse primer andsequenced using an ABI-prism 3130 Genetic Analyzer (AppliedBiosystems).

2.4.2.3. RACE. A rapid amplification of cDNA ends (RACE) strategywas adopted to determine the cDNA sequence of the region flankingthe 30-end of the partial cDNA sequence obtained by degenerate PCR.RACE-ready template was prepared according to the recommenda-tions of the GeneRacer kit (Invitrogen), using 25 ng of the isolatedtotal RNA in a 10 ml reaction. Approximately 1 ml of the RACE-readycDNA reaction servedas template for a 30-RACE reactionusing a gene-specific primer RiAFP-GP1 (50-ATCAACATCAACAGCCACAGCAACAG-30, 0.2 mM) in combinationwith the 30-RACE adapter primer (0.6 mM)that was provided with the kit. The other ingredients in the 25 mlreaction were 1� Buffer J (Invitrogen), MgCl2 (2.5 mM), dNTP’s(2.5 mM, Invitrogen), 2.5 units of GoTaq�Flexi DNA Polymerase(Promega, Madison, USA). The PCR program included an initialdenaturation step at 94 �C (5 min), followed by 35 amplificationcycles at 94 �C (1 min), 60 �C (2 min), 72 �C (2 min), and a finalextension step at 72 �C (10 min). To increase specificity, a nested30-RACE was performed using 1 ml of a 1:10 dilution of the initialRACE-reaction in combination with a nested gene-specific primer

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117112

RiAFP-GP2 (50-GTACCGCCTCAGCCACTTCTAATG-30, 0.2 mM) anda nested 30-RACE adapter primer. The other ingredients and the PCRprogram were identical to the initial 30-RACE reaction describedabove.Agarose gel separationof thenested30-RACE reaction revealeda single strong bandwith an apparent size of 325basepairs. The bandwas purified and sequenced as described above.

2.5. Secondary structure prediction

The complete amino acid sequence of RiAFP was subjected to 12secondary structure prediction algorithms freely available on theinternet. These algorithms predict whether the residue is likely tobe part of a b-strand, a helix or a random coil and 2 of them alsopredict the existence of turns. The following algorithms were used:DSC (King and Sternberg, 1996), GOR 4 (Garnier et al., 1996), HNN(Guermeur, 1997), PHD (Rost and Sander, 1993), PREDATOR(Frishman and Argos, 1997), SIMPA96 (Levin et al., 1986), SOPMA(Geourjon and Deléage, 1995), NNPREDICT (Kneller et al., 1990),PORTER (Pollastri and McLysaght, 2005), SAM T02 (Karplus et al.,2003), JUFO (Meiler and Baker, 2003), SSPRO (Pollastri et al.,2002). Turn regions are only predicted by SOPMA and SAM T02.For ease of interpretation, the percentage of each of the differentstructural types ascribed to each residue was displayed as a stackedbar diagram.

3. Results

The antifreeze activity of RiAFP as a function of the AFPconcentration is compared to the antifreeze activity of some of theother known insect AFPs in Fig. 1. The observed activity shows thatRiAFP is among the most potent AFPs known so far.

Fig. 1. Antifreeze activity as a function of concentration of various insect AFPs. (C):a 8.5 kDa isoform from T. molitor (Marshall et al., 2002). (B): different 9 kDa sprucebudworm isoforms (Doucet et al., 2000; Tyshenko et al., 1997). (;): a high-active12 kDa spruce budworm isoform (Leinala et al., 2002). (6): the 12.8 kDa RiAFP. Verticalbars represent �SD.

3.1. Amino acid sequence determination

In all, 7 partial amino acid sequenceswere successfully obtained,3 by Edman degradation and 4 by de novo sequencing. These partialamino acid sequences accounted for 12,203 Da of the 12,791 DaRiAFP, or 95.4% of its mass. Table 1 lists these sequences and themethods used and the enzyme used to generate the fragment.

Edman degradation of the native RiAFP resulted in a 40-residuesequence that revealed theN-terminalpartof theprotein.Threeof thede novo sequenced fragments, m/z 673.4, 1243.6 and 2655.8, over-lapped with the N-terminal sequence obtained by Edman degrada-tion and confirmed and expanded this known N-terminal part ofRiAFP to 53 residues (Fig. 2). Although the fragment m/z 2655.8showed an almost perfect matchwith the 17 last residues within theEdman sequence, e ATGAGAMWTGTTEPGGT, there was a discrep-ancyat3positions,ATGAGAMASGTSEPGGTe. Thedifferencesat thesepositions towards theendof the40-residue longEdmansequencecanbe ascribed to the increased inaccuracy of the Edmanprocedurewiththe length of the sequenced fragment (Stryer, 1995).

The C-terminal residue of any Lys-C generated fragment origi-nating from the internal part of the sequence should be K, since thisenzyme cuts at this residue. The sequence of the 14-residue Lys-Cfragment m/z 1426.7, SFARNTATTTVTAS (Table 1), does not containK at its terminal end and was therefore identified as the likely C-terminal end of the protein. Lys-C digestion resulted in only 3fragments, m/z 2407.2, 1427.7 and 8987.3 (Fig. 2). The first of thesefragments corresponds to the mass of the N-terminal sequence ofRiAFP ending with K at position 23, whereas the second fragment isthe above mentioned identified C-terminal of RiAFP. The largefragment m/z 8987.3 apparently comprised the entire mid-sectionof RiAFP, since the sum of these masses less 2 water moleculescorresponds closely to the total mass of RiAFP (Fig. 2). The C-terminal of this large fragment should be K and be immediatelyfollowed in the sequence by the identified C-terminal end of RiAFP.Of the remaining fragments listed in Table 1, only the 29-residuesequence of m/z 2812.4 have K at its C-terminal. This fragmenttherefore likely belongs in front of the N-terminal end of the iden-tified C-terminal of RiAFP, extending the constructed C-terminalend of RiAFP to 43 residues (Fig. 2). The position of fragment m/z2971.4 within the sequence (Table 1) could not be identified basedon the available information. This was because approximately 4.6%of the sequencewas not determined de novo, and it was not possibleto determine if this lacking part of the sequence preceded theN-terminal end or followed the C-terminal end of m/z 2971.4.Nevertheless, the identity and position of the first 53 residues of theN-terminal end and the last 43 residues of the C-terminal end of theRiAFP sequence was obtained in this way (Fig. 2).

3.2. DNA sequence determination

The degenerate PCR-procedure generated a 252 nucleotide cDNAsequence product whereas the subsequent 30-RACE procedure

Table 1Amino acid sequences of RiAFP peptide fragments. Numbers preceding the sequencedenote the m/z-value of the fragment. Method of sequencing: a) Edman, b) De novo.Enzyme used prior to sequencing: c) trypsin, d) Lys-C. See on-line material forexamples of mass spectra.

Nativea) YSXRAVGVDGRAVTDIQGTXHAKATGAGAMWTGTTEPGGT673.4ac) AVGVDGR1243.6bd) AVTDIQGTCHAK2655.8bc) ATGAGAMASGTSEPGSTSTATATGRGATAR2971.4bc) GTATTTATGTASATSNAIGQGTATTTATGSAGGR2812.4bd) ATGSATTSSSASQPTQTQTITGPGFQTAK1426.7ad) SFARNTATTTVTAS

Fig. 2. Construction of the RiAFP sequence. The assembling of the peptide fragments, their match to the cDNA products and the location and identity of the missing part of theprotein sequence. Numbers inside the boxes are the m/z-values of the peptide fragments listed in Table 1. Numbers above peptide fragments denote the location of the first residueof that fragment in the complete sequence. The degenerate PCR-product codes residue 3e86 whereas the RACE-product codes residue 69e136 of the protein. The peptide fragmentSTSTGR was deduced from the cDNA sequence only and is located in position 54e59 within the peptide sequence.

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117 113

resulted in a 300 nucleotide cDNA sequence. The 54 nucleotides atthe 50-end of the RACE-product were identical to the 54 nucleotideresidues of the 30-end of the degenerate PCR-product (Table 2). Thetranslated product of the combined cDNA sequence revealed a 134amino acid sequence followed by stop codons and a poly-a tail.Starting at the 30-end of the DNA sequence, the coded amino acidsequence confirmed the last 51 of the constructed 53-residuesequence at the N-terminal part of RiAFP and the 43-residue con-structed sequence at the C-terminal part of RiAFP (see above). TheDNA sequence also confirmed that the C-terminal part of RiAFP endswith e SFARNTATTVAS, as the next codon in the cDNA is a stopsignal. The 34-residue fragmentm/z 2971.4 (Table 1) was identical tothe translated region within the cDNA product that was followed bythat which coded for the constructed 43 amino acid sequence at theC-terminal part of RiAFP, whereas the small part of the amino acidsequence that was missing from the protein sequencing procedureswas identified from the translated cDNA as e STSTGR e and itslocation is between the C-end of the constructed N-terminalsequence and the N-terminal of fragment m/z 2971.4. Theconstruction of the complete amino acid sequence of RiAFP is illus-trated in Fig. 2 and the complete amino acid sequence and the cDNAsequence is aligned in Table 2.

3.3. Sequence characteristics

BLAST searches did not find any protein with significant simi-larity in current databases.

It was knownprior to this study that RiAFP contains only a singledisulfide bond (Kristiansen et al., 2005). Table 2 shows that its twoCys residues are located at position 3 and 20, close to its N-terminus.

Table 2Alignment of the nucleotide- and amino acid sequence of RiAFP. Underlined nucleotides mand 30-RACE, respectively. Underlined amino acid residues mark the missing 6-residue frNucleotide sequence accession number: HQ540314.

tgt agg gcg gtg ggg gtg gac gga cgaY S C R A V G V D G Rcat gca aaa gcg aca ggt gcc ggg gca atg gctH A K A T G A G A M Atca aca gcc aca gca aca gga cgt ggg gcc actS T A T A T G R G A Taca gcc aca acg aca gcc aca ggt acc gcc tcaT A T T T A T G T A Saca gca aca aca aca gcc act gga tct gct ggtT A T T T A T G S A Gtca tcc agc gca tcc cag cca aca caa act caaS S S A S Q P T Q T Qgcg aaa tca ttt gca cga aac act gcc aca accA K S F A R N T A T Taga tac aat tat gag tta cca ttt ttt tat taaaaa aaa aaa aaa aaa aaa aaa aaa

Inspection of the complete 136 amino acid sequence showedthat RiAFP contains 5 internal repeats of the 7-residue patternTxTxTxT and that the residues 19e25 also seems to make upa partial such pattern as TxxxxxT (Table 3). The ‘x’-positions withinthese repeats show a predominance of the small residues A or T,and they tend to be flanked by the small residues G or A. At the N-terminal end, G is preceded by the more bulky residues Q or R.Table 3 shows different parts of the amino acid sequence aligned sothat the different TxTxTxT-motifs are arranged on top of each other.The table suggests that there is a 13-residue consensus repeatsegment A/G1-R/Q2-G3-T4-x5-T6-x7-T8-x9-T10-G11-x12-A/G13 occur-ring six times within the sequence and that each of these repeatsare separated by non-repeat regions varying in length from sevento fourteen residues. The positions of the T-residues in the centralTxTxTxT-part of the pattern are the most conserved, missing onlyfrom the first pattern. G occupies 8 of the 12 flanking positions ofthe central TxTxTxT-motif, corresponding to position 3 and 11 inthe consensus repeat segment. Table 3 also shows that the repeatsegment tends to begin with A or G at position 1 and end with thesame residues at position 13 and that the “x”-residues of the centralTxTxTxT-motif are mostly T or A.

The results of the twelve secondary structure prediction algo-rithms are illustrated in Fig. 3. Overall, these predictions suggestthat the central TxTxTxT-motifs within each repeat have a highpropensity towards b-strand formation. This is also, to a lesserextent, the case for the central parts of the non-repeating regionsbetween the repeats. Most of the ten suggested turn regions arelocated very close to the central TxTxTxT-motifs and all helixcontent is located in the regions between the TxTxTxT-motifs. Also,the proposed helix content increases when moving from theN-terminal towards the C-terminal end of the protein. Overall,

ark the overlapping parts of the nucleotide sequences obtained with degenerate PCRagment of the peptide. Bold letters denote stop codons within the 30-RACE product.

gca gtt act gat atc caa gga act tgt 54A V T D I Q G T C 20tcc ggg acg tct gaa cct gga tca aca 114S G T S E P G S T 40gcc aga tca act tcg aca gga aga gga 174A R S T S T G R G 60gcc act tct aat gcc att ggt cag gga 234A T S N A I G Q G 80ggg agg gca aca gga tcg gca acc aca 294G R A T G S A T T 100acc atc acc gga cca ggc ttt caa acg 354T I T G P G F Q T 120act gtg acg gca tct tag aca taa tcc 414T V T A S 136caa taa agt gac tta aat gaa aaa aaa 474

498

Table 3Alignment of the repeat regions of RiAFP. Numbers denote the positions of theresidues within the sequence starting from the N-terminal.

1e15 Y S C R A V G V D G R A V T D16e36 I Q G T C H A K A T G A G A M A S G T S E37e57 P G S T A T A T A T G R G A T A R S T S T58e77 G R G T A T T T A T G T A S A T S N A I78e104 G Q G T A T T T A T G S A G G R A T G S A T T S S S A105e124 S Q P T Q T Q T I T G P G F Q T A K S F125e136 A R N T A T T T V T A S

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117114

these predictions indicate that the TxTxTxT-motifs form a b-sheeton one side of a coil, as seen in other insect AFPs containing the TxTice-binding motif.

4. Discussion

The sequence of RiAFP contains repeat segments with a TxT-richcentre as seen in AFPs from the beetles T. molitor and D. canadensis.However, these repeats are separated by apparently non-repeatingsegments varying in length from 13 to 20 residues. In addition, thecentral TxTxTxT-motifs of each of the segments of RiAFP areessentially triplets of the TxT-motifs previously found in AFPs fromT. molitor and D. canadensis and in the lepidopteran genus Chori-stoneura. The presence of only a single disulfide bridge in RiAFP asopposed to the high content of such bridges in other insect AFPscontaining the TxT-motif is also noteworthy. These differences, andthe fact that no match was found with any other AFP using BLAST,suggest that this protein represents a new type of AFP.

It is generally assumed that AFPs cause their antifreeze effect byadsorbing onto the ice surface (Raymond and DeVries, 1977;Kristiansen and Zachariassen, 2005) and that this adsorptionevent requires an almost perfect structural match between the ice-binding residues within the ice-binding site of the AFP and thearrangement of oxygen atoms at the ice surface. The stack of 5e8TxT-motifs that makes up the ice-binding site within the AFPs fromboth T. molitor and C. fumiferana forms a b-sheet (Liou et al., 2000b;Graether et al., 2000). The 4.75 Å inter-strand spacing of b-sheets fitstructurally quite well with the intermolecular spacing of oxygenatoms in ice, allowing the oxygen atoms of the exposed OH-groupsof the T-residues within the TxT-stack, together with those ofexternally bound water molecules, to match the pattern of oxygenatoms within both the basal- and prism planes of ice (Graetheret al., 2000).

The predicted structure of the TxTxTxT-motifs within RiAFPtends towards b-strand configuration (Fig. 3). This is also the case toa lesser degree for the central parts of the intermediate sections

Fig. 3. Predicted secondary structure of RiAFP. The stacked bar shows the percentage of seEach algorithm only assigns a single structure to each residue. Black: b-strand, dark grey: hethe six tentative TxTxTxT ice-binding motifs are illustrated above the diagram as white boxlocation of the three P-residues within the structure. A one-letter code of the structure pre

between these motifs. Thus, taken together with the structuralinsight from other insect AFPs containing TxT-motifs, thesepredictions indicate that the 6 TxTxTxT-motifs within RiAFP forma b-sheet that makes up its ice-binding site on one side of a coiledstructure and that the opposing side of the coil also containsa significant portion of b sheet formation. It is noteworthy that allregions predicted to tend towards helix content lie outside theregions containing the TxTxTxT-motifs and that most of the pre-dicted turn regions within the structure are located at the edges ofthe predicted b-stand regions. Within the structure, 8 of the 12flanking positions of the 6 different TxTxTxT-motifs of RiAFP areoccupied by glycine (G, Table 3). This residue has a high propensityin turn regions of proteins due to its single hydrogen side chain thatallows the backbone of proteins to be quite flexible at these posi-tions (Stryer, 1995). It is also significant that 2 of the 3 proline foundwithin the structure of RiAFP, P107 and P116, are positioned close toone of the TxTxTxT-motifs, P107 occupying a flanking position. Theattachment of the side chain of P to the backbone nitrogen causesthe polypeptide backbone to bend (Stryer,1995). The predicted turnregions and the location of G, P and other small residues at thesepositions throughout the RiAFP sequence indicates that thesecondary structure of RiAFP undergoes an abrupt change in theorientation of the polypeptide backbone at or close to the flankingpositions of the TxTxTxT-motifs.

With their 12 or 15 residues per turn, the b-helical AFPs fromT. molitor and C. fumiferana are remarkably compact compared tothe 18e22 residues per turn seen in non-antifreeze b-helicalproteins (Graether and Sykes, 2004). This structural tightness ismade possible partly by the many disulfide bonds throughout thestructure of the AFPs. In the AFPs from the beetles T. molitor andD. canadensis, cysteine is the central residue of the TxT-motifs, eachforming a disulfide bond with a C located at the opposite side of thecoil (Li et al., 1998b; Liou et al., 2000a,b). In the AFPs of moth sprucebudworm the positions of the Cys residues are not repetitive and,although the disulfide bonds cross the coiled structure, they are notaligned as ordered as in the AFPs of the beetles T. molitor andD. canadensis (Gauthier et al., 1998; Graether et al., 2000). We havepreviously shown that RiAFP contains only a single disulfide bond(Kristiansen et al., 2005), and Table 2 shows that its two Cys resi-dues are located at position 3 and 20, close to its N-terminus.Although the primary sequence of RiAFP lends support to thenotion of a coiled configuration, the absence of multiple centraldisulfide bonds and the number of residues separating eachTxTxTxT-motif suggests that RiAFP would not have as tight foldingas seen in other insect AFPs. A coiled configuration with theTxTxTxT-motifs aligned on one side of the coil would imply that theloops within RiAFP would vary in length from 20 to 27 residues.Such larger coiled structures have been proposed for the AFP-like

condary structure predicted by 12 different secondary structure prediction algorithms.lix, light grey: random coil, white: turn region. See text for references. The locations ofes. Also illustrated is the disulfide bond (SeS) located close to the N-terminal and thedictions is available as on-line material.

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117 115

ice recrystallisation inhibition agents of the plants perennialryegrass, Lolium perenne (Kuiper et al., 2001) and carrot, Daucuscarrota (Zhang et al., 2004) and also that of the Antarctic bacterium,Marinomonas primoryensis (Garnham et al., 2008). In these modelsthe proposed loops consist of 15, 24 and 19 residues, respectively.The ice recrystallisation inhibition agent of D. carrota contains 2disulfide bridges close to its N-terminal, whereas those of the othertwo species do not contain cysteine. All of these models rely onstructural stability provided by a hydrophobic core predominantlyconsisting of Val, Leu and Ile. Notable hydrophobic residues inRiAFP are 26 A (20%), 4 V and 3 I. However, it is also rich inpotentially hydrogen-bonding residues, since it contains 13 T,excluding those of the TxTxTxT-motifs, and 17 S.

The central ‘x’-residue in the TxT-motifs of T. molitor andC. fumiferana have been shown to face into the interior of the coiledstructure, a prerequisite for the required intimate contact betweenthe OH-groups of the T-residues within the TxT-motif and ice forproper adsorption to develop. In the case of T. molitor and D. can-adensis this central position is occupied by C, whereas in C. fumi-ferana C, A and T predominate. In RiAFP, 9 of the 18 corresponding‘x’-positions in the 6 TxTxTxT-motifs are occupied by A and 3 by T.However, the TxTxTxT-motif spanning residues 108e114 seem oddin this respect in that the central ‘x’-positions are occupied by Gluand Ile, residues with large side-chains that could potentially bestructurally disruptive if oriented into the core of a coiled structure.It is noteworthy that this TxTxTxT-motif is separated from thepreceding motif by a segment 6e7 residues longer than the cor-responding segments, which would result in a larger loop thatcould potentially fulfil the extra spatial requirement of these largerside-chains. In addition, two of the three P found within thesequence, P107 and P116, are positioned immediately adjacent to theTxTxTxT-motif containing the bulky side-chains. These P-residuescould potentially cause the loop of this segment to be more abrupt.

Fig. 1 shows that at equimolar concentrations RiAFP has a verystrong ability to evoke antifreeze activity compared to most otherinsect AFPs. It would be tempting to ascribe this strong capacity tothe presence of more ice-binding residues within the expandedTxTxTxT-motifs of RiAFP, since the ice-binding sites of the otherspecies consist of stacks of the shorter TxT-motifs. Indeed, severalinvestigators have reported that increased number of TxT-motifswithin the ice-binding site of other insect AFPs increases theresultant antifreeze activity (Leinala et al., 2002; Marshall et al.,2004). An alternative approach to explain the differences in theantifreeze potency of insect AFPs is based on their solubility in thewater phase surrounding the ice crystal (Kristiansen andZachariassen, 2005; Kristiansen et al., 2008). This explanation isbased on the fact that while the ice crystal is at the equilibriummelting temperature, the ice lattice and the water phase is sepa-rated by a chaotic transition zone that extends 1e2 nm in depth.According to this view, as long as the ice crystal remains at theequilibrium melting temperature this iceewater interfacial regiondoes not provide any clear ice surface for the AFPs to adsorb ontoand so the AFPs can only obtain a steady-state equilibrium distri-bution between the bulk water phase and the iceewater interfacialregion. At the instant the crystal is cooled the situation changes, asthe AFPs within the iceewater surface region adsorb irreversiblyonto the solidifying iceewater surface region. Thus, this approachascribes the antifreeze effect solely to those AFP molecules that arewithin the interfacial region at the instant of cooling. Since a low-ered AFP solubility in the bulk water phase should result in a largerfraction of the AFPs accumulating within the interfacial region priorto cooling this implies that lowered AFP solubility should also resultin increased antifreeze activity.

Evidence that the solubility of the AFPs contributes to theirantifreeze potency comes from several sources. Salts have been

shown to enhance antifreeze activity (Li et al., 1998c; Evans et al.,2007; Kristiansen et al., 2008; Wang et al., 2009). Kristiansenet al. (2008) showed that the ranking of salts as enhancers ofantifreeze activity of RiAFP corresponded to the Hofmeister series,the ranking of salts according to their ability to lower proteinsolubility. Further, the magnitude of the salt-induced enhancementof antifreeze activity corresponded quantitatively to the effects ofthese salts on the solubility of a protein the size of RiAFP. These andother relations presented in that study strongly suggest that saltsact as enhancers of antifreeze activity by lowering the solubility ofthe antifreeze protein in solution. More recently, Wang et al. (2009)also found that salts that enhanced the antifreeze activity of anantifreeze protein from the beetle D. canadensis ranked similar tothe Hofmeister series.

The ice-binding sites of AFPs have been shown to be morehydrophobic than the rest of the AFP structure (Sönnichsen et al.,1996; Chao et al., 1997; Baardsnes and Davies, 2002). Thus, iso-forms with larger ice-binding sites could be expected to be lesssoluble than isoforms with smaller ice-binding sites. According tothe above explanation, larger ice-binding sites should thereforeindirectly result in a more potent AFP, by causing the protein to beless soluble. Thus, the apparent link between the size of the ice-binding sites of AFPs and their potency may be less direct thanpreviously thought and arise from a bulk physiochemical propertyof these molecules, i.e. their solubility within the water phase,which simply varies with the size of their ice-binding sites. Inaddition, the more potent isoforms with larger ice-binding sites arealso overall larger structures than the less potent isoforms withsmaller ice-binding sites. For structurally similar compounds theirsolubility tends to decrease with increased size (Flory, 1953; vanOss et al., 1986). In this respect the 12.8 kDa RiAFP is relativelylarge compared to most other insect AFPs, which are mostly in thesize-range of 7e10 kDa.

As is the case for the structural basis of their antifreeze potency,little is known of the events and processes that drove the evolutionof insect AFPs. The coleopteran and lepidopteran species studied todate all express a number of AFP isoforms within their body fluids,and we have previously reported that several isoforms of RiAFP arepresent in the hemolymph of R. inquisitor (Kristiansen et al., 2005).The diversity seen among the many AFP isoforms of T. molitor andD. canadensis have been suggested to result from a selective pres-sure towards adaptive diversity at the amino acid level (Swansonand Aquadro, 2002) or, alternatively, that the selective pressurehas rather been towards a higher AT content at the nucleotide leveland that the resultant amino acid substitutions are functionallyneutral (Graham et al., 2007). It was argued in the latter case thatthe increased AT-level may facilitate transcription or translation atlower temperatures.

The similarity between AFPs of several closely related species ofthe Lepidopteran genus Choristoneura suggests that they emergedprior to species divergence about 3.2e3.7 Mya, corresponding tothe cold period preceding the Pleistocene ice ages that started some3 Mya (Tyshenko et al., 2005). The Coleopterans T. molitor andD. canadensis belong to two different families, the Tenebrionidaeand the Pyrochroidae, respectively, and are obviouslymore distantlyrelated than those of the genus Choristoneura. Nonetheless, theirshared consensus 12-residue sequence TCTxSxxCxxAx indicatesthat they may also have a common genetic origin (Graham et al.,2007). Such structural similarity may however also have comeabout by convergent evolution, as is the case for several of thestructurally similar forms of fish AFPs present in distantly relatedspecies. This is perhaps best illustrated by the presence of struc-turally similar antifreeze glycoproteins (AFGPs) in as distantlyrelated species as Antarctic notothenoids and Arctic cods. TheAFGPs of the Antarctic notothenoids probably developed in

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117116

a common notothenoid ancestor about 10e15 Mya, at a time whenthe cooling of the Antarctic caused freezing water temperatures,and long after cods and nototenoids had branched into differentgroups (Bargelloni et al., 1994; Chen et al., 1997a,b).

Both of the beetles T. molitor and D. canadensis belong to thesuperfamily Tenebrionoidea whereas R. inquisitor belongs toa different superfamily, the Chrysomeloidea. The low similaritybetween these structures and RiAFP and the failure to find anymatch with known sequences using BLAST suggest that RiAFP hasa different genetic origin than that of the AFPs from T. molitor andD. canadensis. It has been suggested that the TxT-motif is an optimalbuilding block to achieve a structural match to ice (Graether et al.,2000). The convergent evolution of this ice-binding motif amongsuch diverse species as Coleopterans and Lepidopterans and itsoccurrence in an expanded form in RiAFP testifies to this view.

5. Conclusion

This study reports on the primary sequence of a 12.8 kDa RiAFPpurified from the hemolymph of the longhorn beetle R. inquisitor,and is the first report to provide structural information on an AFPfrom the superfamily Chrysomeloidea. Information is only availablefrom two other species of beetle, D. canadensis and T. molitor, whichbelongs to the superfamily Tenebrionoidea. These previously char-acterized AFPs are both constructed as a series of repeat units, eachunit being of 12 or 13 residues in length. Although the sequence ofRiAFP contains 6 repeat regions of 13 residues, these regions arespaced apart by non-repeat segments of varying length. The otherknown AFPs from beetles contain several “TxT” ice-binding motifsat regular intervals. The repeat regions of RiAFP contain theexpanded TxTxTxT-form of this motif at the centre of its repeatregions. RiAFP only contains a single disulfide bridge, located closeto its N-terminal, as opposed to themultiple disulfide bridges foundthroughout the sequence of other known beetle AFPs, and suggeststhat it does not have as tight folding as those of other insect AFPswith multiple disulfide bridges.

Acknowledgement

We thank MSc Nhon Van Nguyen at the Department ofBiochemistry and Molecular Biology, University of SouthernDenmark for de novo sequencing and the Proteomic node atDepartment of Cancer Research and Molecular Medicine at NTNUfor helpful discussions and comments.

Appendix. Supplementary material

Supplementary material related to this article can be foundonline at doi:10.1016/j.ibmb.2010.11.002.

References

Andorfer, C.A., Duman, J.G., 2000. Isolation and characterization of cDNA clonesencoding antifreeze proteins of the pyrochroid beetle Dendroides canadensis.J. Insect Physiol. 46, 365e372.

Baardsnes, J., Davies, P.L., 2002. Contribution of hydrophobic residues to ice bindingby fish type III antifreeze protein. Biochim. Biophys. Acta 1601, 49e54.

Bargelloni, L., Ritchie, P.A., Patarnello, T., Battaglia, B., Lambert, D.M., Meyer, A., 1994.Molecular evolution at subzero temperatures: mitochondrial and nuclearphylogenies of fishes from Antarctica (Suborder Notothenioidei) and theevolution of antifreeze glycopeptides. Mol. Biol. Evol. 11, 854e863.

Beardsley, R.L., Reilly, J.P., 2002. Optimization of guanidination procedures forMALDI Mass Mapping. Anal. Chem. 74, 1884e1890.

Chao, H., Houston Jr., M.E., Hodges, R.S., Kay, C.M., Sykes, B.D., Loewen, M.C.,Davies, P.L., Sönnichsen, F.D., 1997. A diminished role for hydrogen bonds inantifreeze protein binding to ice. Biochemistry 36, 14652e14660.

Chen, L., DeVries, A.L., Cheng, C.-H.C., 1997a. Evolution of antifreeze glycoproteingene from a trypsinogen gene in Antarctic notothenioid fish. Proc. Natl. Acad.Sci. U.S.A. 94, 3811e3816.

Chen, L., DeVries, A.L., Cheng, C.-H.C., 1997b. Convergent evolution of antifreezeglycoproteins in Antarctic notothenioid fish and Arctic cod. Proc. Natl. Acad. Sci.U.S.A. 94, 3817e3822.

Doucet, D., Tyshenko, M.G., Kuiper, M.J., Graether, S.P., Sykes, B.D., Daugulis, A.J.,Davies, P.L., Walker, V.K., 2000. Structure-function relationships in sprucebudworm antifreeze protein revealed by isoform diversity. Eur. J. Biochem. 267,6082e6088.

Doucet, D., Tyshenko, M.G., Davies, P.L., Walker, V.K., 2002. A family of expressedantifreeze protein genes from the moth, Choristoneura fumiferana. Eur. J. Bio-chem. 269, 38e46.

Duman, J.G., 2002. The inhibition of ice nucleators by insect antifreeze proteins isenhanced by glycerol and citrate. J. Comp. Physiol. B 172, 163e168.

Duman, J.G., Bennett, V., Sformo, T., Hochstrasser, R., Barnes, B.M., 2004. Antifreezeproteins in Alaskan insects and spiders. J. Insect Physiol. 50, 259e266.

Evans, P.E., Hobbs, R.S., Goddard, S.V., Fletcher, G.L., 2007. The importance of dis-solved salts to the in vivo efficacy of antifreeze proteins. Comp. Biochem.Physiol. A 148, 556e561.

Flory, P.J., 1953. Principles of Polymer Chemistry. Cornell University Press, London.Frishman, D., Argos, P., 1997. 75% accuracy in protein secondary structure prediction.

Proteins 27, 329e335.Garnham, C.P., Gilbert, J.A., Hartman, C.P., Campbell, R.L., Laybourn-Parry, J.,

Davies, P.L., 2008. A Ca2þ-dependent bacterial antifreeze protein domain hasa novel b-helical ice-binding fold. Biochem. J. 411, 171e180.

Garnier, J., Gibrat, J.-F., Robson, B., 1996. GOR secondary structure predictionmethod version IV. Meth. Enzymol. 266, 540e553.

Gauthier, S.Y., Kay, C.M., Sykes, B.D., Walker, V.K., Davies, P.L., 1998. Disulfide bondmapping and structural characterization of spruce budworm antifreeze protein.Eur. J. Biochem. 258, 445e453.

Geourjon, C., Deléage, G., 1995. SOPMA: significant improvement in proteinsecondary structure prediction by consensus prediction from multiple align-ments. Bioinformatics 11, 681e684.

Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R., Roepstorff, P., 1999. Samplepurification and preparation technique based on nano-scale reversed-phasecolumns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J. Mass Spectrom. 34,105e116.

Graether, S.P., Sykes, B.D., 2004. Cold survival in freeze-intolerant insects, thestructure and function of b-helical antifreeze proteins. Eur. J. Biochem. 271,3285e3296.

Graether, S.P., Kuiper, M.J., Gagnè, S.M., Walker, V.K., Jia, Z., Sykes, B.D., Davies, P.L.,2000. b-helix structure and ice-binding properties of a hyperactive antifreezeprotein from an insect. Nature 406, 325e328.

Graham, L.A., Davies, P.L., 2005. Glycine-rich antifreeze proteins from snow fleas.Science 310, 461.

Graham, L.A., Qin, W., Lougheed, S.C., Davies, P.L., Walker, V.K., 2007. Evolution ofhyperactive, repetitive antifreeze proteins in beetles. J. Mol. Evol. 64, 387e398.

Guermeur, Y., 1997. Combinaison de classifieurs statistiques, Application a laprediction de structure secondaire des proteines. PhD thesis, Université Paris 6.

Jia, Z., Davies, P.L., 2002. Antifreeze proteins: an unusual receptoreligand interac-tion. Trends Biochem. Sci. 27, 101e106.

Karplus, K., Karchin, R., Draper, J., Casper, J., Mandel-Gutfreund, Y., Diekhans, M.,Hughey, R., 2003. Combining local-structure, fold-recognition, and new-foldmethods for protein structure prediction. Proteins 53, 491e496.

King, R.D., Sternberg, M.J.E., 1996. Identification and application of the conceptsimportant for accurate and reliable protein secondary structure prediction.Protein Sci. 5, 2298e2310.

Kneller, D.G., Cohen, F.E., Langridge, R., 1990. Improvements in protein secondarystructure prediction by an enhanced neural network. J. Mol. Biol. 214, 171e182.

Kristiansen, E., Zachariassen, K.E., 2005. The mechanism by which fish antifreezeproteins cause thermal hysteresis. Cryobiology 51, 262e280.

Kristiansen, E., Pedersen, S., Ramløv, H., Zachariassen, K.E., 1999. Antifreeze activityin the cerambycid beetle Rhagium inquisitor. J. Comp. Physiol. B 169, 55e60.

Kristiansen, E., Ramløv, H., Hagen, L., Pedersen, S.A., Andersen, R.A., Zachariassen, K.E.,2005. Isolation and characterization of hemolymphantifreezeproteins from larvaeof the longhorn beetle Rhagium inquisitor (L.). Comp. Biochem. Physiol. B 142,90e97.

Kristiansen, E., Pedersen, S.A., Zachariassen, K.E., 2008. Salt-induced enhancementof antifreeze protein activity: a salting-out effect. Cryobiology 57, 122e129.

Kuiper, M.J., Davies, P.L., Walker, V.K., 2001. A theoretical model of a plant antifreezeprotein from Lolium perenne. Biophys. J. 81, 3560e3565.

Leinala, E.K., Davies, P.L., Doucet, D., Tyshenko, M.G., Walker, V.K., Jia, Z., 2002.A b-helical antifreeze protein isoform with increased activity. J. Biol. Chem. 227,33349e33352.

Levin, J.M., Robson, B., Garnier, J., 1986. An algorithm for secondary structure deter-mination in proteins based on sequence similarity. FEBS Lett. 205, 303e308.

Li, N., Kendrick, B.S., Manning, M.C., Carpenter, J.F., Duman, J.G., 1998a. Secondarystructure of antifreeze proteins from overwintering larvae of the beetle Den-droides canadensis. Arch. Biochem. Biophys. 360, 25e32.

Li, N., Chibber, B.A.K., Castellino, F.J., Duman, J.G., 1998b. Mapping of disulfidebridges in antifreeze proteins from overwintering larvae of the beetle Den-droides canadensis. Biochemistry 37, 6343e6350.

Li, N., Andorfer, C.A., Duman, J.G., 1998c. Enhancement of insect antifreeze proteinactivity by solutes of low molecular mass. J. Exp. Biol. 201, 2243e2251.

Lin, F.-H., Graham, L.A., Campbell, R.L., Davies, P.L., 2007. Structural modeling ofsnow flea antifreeze protein. Biophys. J. 92, 1717e1723.

E. Kristiansen et al. / Insect Biochemistry and Molecular Biology 41 (2011) 109e117 117

Liou, Y.-C., Thibault, P., Walker, V.K., Davies, P.L., Graham, L.A., 1999. A complexfamily of highly heterogeneous and internally repetitive hyperactive anti-freeze proteins from the beetle Tenebrio molitor. Biochemistry 38,11415e11424.

Liou, Y.-C., Daley, M.E., Graham, L.A., Kay, C.M., Walker, V.K., Sykes, B.D.,Davies, P.L., 2000a. Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria. Protein Expr. Purif. 19,148e157.

Liou, Y.-C., Tocilj, A., Davies, P.L., Jia, Z., 2000b. Mimicry of ice structure by surfacehydroxyls and water of a b-helix antifreeze protein. Nature 406, 322e324.

Marshall, C.B., Daley, M.E., Graham, L.A., Sykes, B.D., Davies, P.L., 2002. Identificationof the ice-binding face of antifreeze protein from Tenebrio molitor. FEBS Lett.529, 261e267.

Marshall, C.B., Daley, M.E., Sykes, B.D., Davies, P.L., 2004. Enhancing the activity ofa b-helical antifreeze protein by the engineered addition of coils. Biochemistry43, 11637e11646.

Meiler, J., Baker, D., 2003. Coupled prediction of protein secondary and tertiaryStructure. Proc. Natl. Acad. Sci. U.S.A. 100, 12105e12110.

Olsen, T.M., Duman, J.G., 1997. Maintenance of the supercooled state in the gut fluidof overwintering pyrochroid beetle larvae, Dendroides canadensis: role of icenucleators and antifreeze proteins. J. Comp. Physiol. B 167, 114e122.

Olsen, T.M., Sass, S.J., Li, N., Duman, J.G., 1998. Factors contributing to seasonalincreases in inoculative freezing resistance in overwintering fire-colored beetlelarvae Dendroides canandensis (Pyrochroidae). J. Exp. Biol. 201, 1585e1594.

van Oss, C.J., Good, R.J., Chaudhury, M.K., 1986. Solubility of proteins. J. ProteinChem. 5, 385e405.

Pentelute, B.L., Gates, Z.P., Tereshko, V., Dashnau, J.L., Vanderkooi, J.M.,Kossiakoff, A.A., Kent, S.B.H., 2008. X-ray structure of snow flea antifreezeprotein determined by racemic crystallization of synthetic protein enantiomers.J. Am. Chem. Soc. 130, 9695e9701.

Pollastri, G., McLysaght, A., 2005. Porter: a new, accurate server for proteinsecondary structure prediction. Bioinformatics 21, 1719e1720.

Pollastri, G., Przybylski, D., Rost, B., Baldi, P., 2002. Improving the prediction ofprotein secondary structure in three and eight classes using recurrent neuralnetworks and profiles. Proteins 47, 228e235.

Raymond, J.A., DeVries, A.L., 1977. Adsorption inhibition as a mechanism of freezingresistance in polar fishes. Proc. Natl. Acad. Sci. U.S.A. 74, 2589e2593.

Rost, B., Sander, C., 1993. Prediction of protein secondary structure at better than70% accuracy. J. Mol. Biol. 232, 584e599.

Sönnichsen, F.D., DeLuca, C.I., Davies, P.L., Sykes, B.D., 1996. Refined solutionstructure of type III antifreeze protein: hydrophobic groups may be involved inthe energetics of the proteineice interaction. Structure 4, 1325e1337.

Stryer, L., 1995. Biochemistry, fourth ed. W.H. Freeman and Company, New York.Swanson, W.J., Aquadro, C.F., 2002. Positive darwinian selection promotes hetero-

geneity among members of the antifreeze protein multigene family. J. Mol. Evol.54, 403e410.

Thomson, W., 1871. On the equilibrium of vapour at a curved surface of liquid.Philos. Mag. 42, 448e452.

Tyshenko, M.G., Doucet, D., Davies, P.L., Walker, V.K., 1997. The antifreeze potential ofthe spruce budworm thermal hysteresis protein. Nat. Biotechnol. 15, 887e890.

Tyshenko, M.G., Doucet, D., Walker, V.K., 2005. Analysis of antifreeze proteinswithin spruce budworm sister species. Insect Mol. Biol. 14, 319e326.

Wang, S., Amornwittawat, N., Banatlao, J., Chung, M., Kao, Y., Wen, X., 2009. Hof-meister effects of common monovalent salts on the beetle antifreeze proteinactivity. J. Phys. Chem. B 113, 13891e13894.

Zachariassen, K.E., Husby, J.A., 1982. Antifreeze effect of thermal hysteresis agentsprotects highly supercooled insects. Nature 298, 865e867.

Zachariassen, K.E., Li, N.G., Laugsand, A.E., Kristiansen, E., Pedersen, S.A., 2008. Is thestrategy for cold hardiness in insects determined by their water balance? Astudy on two closely related families of beetles: Cerambycidae and Chrys-omelidae. J. Comp. Physiol. B 178, 977e984.

Zhang, D.-Q., Liu, B., Feng, D.-R., He, Y.-M., Wang, S.-Q., Wang, H.-B., Wang, J.-F.,2004. Significance of conservative asparagine residues in the thermal hysteresisactivity of carrot antifreeze protein. Biochem. J. 377, 589e595.