Antifreeze Protein Dimer

A naturally occurring tandem duplication of the 7-kDa type III antifreeze protein from Antarctic eel pout (Lycodichthys dearborni) is twice as active as the monomer in depressing the freezing point of a solution. We have investigated the basis for this enhanced activity by producing recombinant analogues of the linked dimer that assess the effects of protein size and the number and area of the ice-binding site(s). The recombinant dimer connected by a peptide linker had twice the activity of the monomer. When one of the two ice-binding sites was inactivated by site-directed mutagenesis, the linked dimer was only 1.2 times more effective than the monomer. When the two monomers were linked through a C-terminal disulfide bond in such a way that their two ice-binding sites were opposite each other and unable to engage the same ice surface simultaneously, the dimer was again only 1.2 times as active as the monomer. We conclude from these analyses that the enhanced activity of the dimer stems from the two ice-binding sites being able to engage to ice at the same time, effectively doubling the area of the ice-binding site. A naturally occurring tandem duplication of the 7-kDa type III antifreeze protein from Antarctic eel pout (Lycodichthys dearborni) is twice as active as the monomer in depressing the freezing point of a solution. We have investigated the basis for this enhanced activity by producing recombinant analogues of the linked dimer that assess the effects of protein size and the number and area of the ice-binding site(s). The recombinant dimer connected by a peptide linker had twice the activity of the monomer. When one of the two ice-binding sites was inactivated by site-directed mutagenesis, the linked dimer was only 1.2 times more effective than the monomer. When the two monomers were linked through a C-terminal disulfide bond in such a way that their two ice-binding sites were opposite each other and unable to engage the same ice surface simultaneously, the dimer was again only 1.2 times as active as the monomer. We conclude from these analyses that the enhanced activity of the dimer stems from the two ice-binding sites being able to engage to ice at the same time, effectively doubling the area of the ice-binding site. Fish are protected from freezing by antifreeze proteins (AFPs), 1The abbreviations used are: AFP, antifreeze protein; wlwAFP, wild type-linker-wild type AFP; wlxAFP, wild type-linker-knockout AFP; wsswAFP, disulfide-linked wild type AFP homodimer; FPLC, fast performance liquid chromatography; HPLC, high performance liquid chromatography; DTT, dithiothreitol. which bind to the surface of nucleating ice crystals in their body fluids, thereby reducing their freezing point below that of the ocean (1Raymond J.A. DeVries A.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2589-2593Crossref PubMed Scopus (676) Google Scholar, 2DeVries A.L. Annu. Rev. Physiol. 1983; 45: 245-260Crossref PubMed Scopus (179) Google Scholar, 3Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-618Crossref PubMed Scopus (468) Google Scholar). AFPs create a local curvature of the ice between adsorbed AFPs, which makes it energetically unfavorable for liquid water to join the ice surface (4Knight C.A. Cheng C.C. DeVries A.L. Biophys. J. 1991; 59: 409-418Abstract Full Text PDF PubMed Scopus (463) Google Scholar). This process, known as the Kelvin effect, produces a non-equilibrium reduction of the freezing point of ice below the melting point (5Wilson P.W. Cryo Lett. 1993; 14: 31-36Google Scholar). The difference between the melting and freezing points is the thermal hysteresis gap, and it is within this temperature range that ice growth is prevented and fish are protected from freezing. Type III AFPs belong to one of several structurally distinct antifreeze protein families found in fishes (6Davies P.L. Sykes B.D. Curr. Opin. Struct. Biol. 1997; 7: 828-834Crossref PubMed Scopus (188) Google Scholar, 7Ewart K.V. Lin Q. Hew C.L. Cell. Mol. Life. Sci. 1999; 55: 271-283Crossref PubMed Scopus (214) Google Scholar, 8Fletcher G.L. Hew C.L. Davies P.L. Annu. Rev. Physiol. 2001; 63: 359-390Crossref PubMed Scopus (400) Google Scholar). These 7-kDa proteins have a compact β-stranded structure (9Antson A.A. Smith D.J. Roper D.I. Lewis S. Caves L.S. Verma C.S. Buckley S.L. Lillford P.J. Hubbard R.E. J. Mol. Biol. 2001; 305: 875-889Crossref PubMed Scopus (106) Google Scholar, 10Sonnichsen F.D. DeLuca C.I. Davies P.L. Sykes B.D. Structure. 1996; 4: 1325-1337Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 11Jia Z. DeLuca C.I. Chao H. Davies P.L. Nature. 1996; 384: 285-288Crossref PubMed Scopus (222) Google Scholar, 12Yang D.S. Hon W.C. Bubanko S. Xue Y. Seetharaman J. Hew C.L. Sicheri F. Biophys. J. 1998; 74: 2142-2151Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Ko T.P. Robinson H. Gao Y.G. Cheng C.H. DeVries A.L. Wang A.H. Biophys. J. 2003; 84: 1228-1237Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) that shows homology to the C-terminal domain of sialic acid synthase (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Structure-function studies have localized the ice-binding residues to a flat, amphipathic surface on the protein (11Jia Z. DeLuca C.I. Chao H. Davies P.L. Nature. 1996; 384: 285-288Crossref PubMed Scopus (222) Google Scholar, 15DeLuca C.I. Chao H. Sonnichsen F.D. Sykes B.D. Davies P.L. Biophys. J. 1996; 71: 2346-2355Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 16Chao H. Sonnichsen F.D. DeLuca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar). This ice-binding face includes several conserved hydrophilic residues (Gln9, Asn14, Thr15, Thr18, Gln44) that potentially form hydrogen bonds with water molecules on the ice surface (10Sonnichsen F.D. DeLuca C.I. Davies P.L. Sykes B.D. Structure. 1996; 4: 1325-1337Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17Jia Z. DeLuca C.I. Davies P.L. Protein Sci. 1995; 4: 1236-1238Crossref PubMed Scopus (18) Google Scholar), flanked by hydrophobic residues (Leu10, Ile13, Leu19, Val20, Val41) on the periphery. Altogether, these residues are thought to make favorable van der Waals contacts with the ice surface (10Sonnichsen F.D. DeLuca C.I. Davies P.L. Sykes B.D. Structure. 1996; 4: 1325-1337Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 12Yang D.S. Hon W.C. Bubanko S. Xue Y. Seetharaman J. Hew C.L. Sicheri F. Biophys. J. 1998; 74: 2142-2151Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 18Baardsnes J. Davies P.L. Biochim. Biophys. Acta. 2002; 1601: 49-54Crossref PubMed Scopus (74) Google Scholar, 19Graether S.P. DeLuca C.I. Baardsnes J. Hill G.A. Davies P.L. Jia Z. J. Biol. Chem. 1999; 274: 11842-11847Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 20Chen G. Jia Z. Biophys. J. 1999; 77: 1602-1608Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition, there may also be stabilizing entropic effects from bringing this somewhat hydrophobic surface into contact with ice (10Sonnichsen F.D. DeLuca C.I. Davies P.L. Sykes B.D. Structure. 1996; 4: 1325-1337Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 21Harding M.M. Ward L.G. Haymet A.D. Eur. J. Biochem. 1999; 264: 653-665Crossref PubMed Scopus (161) Google Scholar). Type III AFP was first reported to bind to the primary prism plane {10-10} of ice (22Cheng C.C. DeVries A.L. di Prisco G. Life Under Extreme Conditions. Springer-Verlag, Berlin1991: 1-14Crossref Google Scholar). However, Antson et al. have recently shown it can bind to several planes lying parallel with or at an acute angle to the c-axis of the ice crystal (9Antson A.A. Smith D.J. Roper D.I. Lewis S. Caves L.S. Verma C.S. Buckley S.L. Lillford P.J. Hubbard R.E. J. Mol. Biol. 2001; 305: 875-889Crossref PubMed Scopus (106) Google Scholar). Approximately 20 type III isoforms from five species of zoarcid fishes have now been sequenced, with an overall 50% sequence identity. One isoform (designated RD3) from the Antarctic eel pout (Lycodichthys dearborni) has two type III AFPs joined in tandem by a nine-amino acid linker peptide (23Wang X. DeVries A.L. Cheng C.H. Biochim. Biophys. Acta. 1995; 1247: 163-172Crossref PubMed Scopus (49) Google Scholar). This duplicated AFP with two similar AFP domains has been referred to in the literature as an intramolecular dimer (24Miura K. Ohgiya S. Hoshino T. Nemoto N. Suetake T. Miura A. Spyracopoulos L. Kondo H. Tsuda S. J. Biol. Chem. 2001; 276: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) although there is no suggestion that the tandemly repeated AFPs contact each other. NMR analysis of the 14.7-kDa RD3 isoform indicates that the linker region is fairly flexible and may allow both ice-binding faces to engage the ice surface at the same time (24Miura K. Ohgiya S. Hoshino T. Nemoto N. Suetake T. Miura A. Spyracopoulos L. Kondo H. Tsuda S. J. Biol. Chem. 2001; 276: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). RD3 was reported to have twice the molar activity of the monomeric isoforms RD1 and RD2 (23Wang X. DeVries A.L. Cheng C.H. Biochim. Biophys. Acta. 1995; 1247: 163-172Crossref PubMed Scopus (49) Google Scholar) and anywhere from 1.5 to six times the activity of the recombinant N-terminal domain alone depending on the concentration tested (24Miura K. Ohgiya S. Hoshino T. Nemoto N. Suetake T. Miura A. Spyracopoulos L. Kondo H. Tsuda S. J. Biol. Chem. 2001; 276: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Biosynthetic trimers and tetramers of type III AFP show a further modest increase in activity (25Nishimiya Y. Ohgiya S. Tsuda S. J. Biol. Chem. 2003; 278: 32307-32312Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Here we have investigated the basis for the increased activity of a recombinant type III AFP dimer modeled on the RD3 isoform. The dimer was made using two identical copies of the Macrozoarces americanus HPLC-12 isoform derivative (Swiss Protein ID P19614). Currently, this is the best characterized type III AFP isoform. Its 1.15-Å x-ray structure has been solved independently by two groups (9Antson A.A. Smith D.J. Roper D.I. Lewis S. Caves L.S. Verma C.S. Buckley S.L. Lillford P.J. Hubbard R.E. J. Mol. Biol. 2001; 305: 875-889Crossref PubMed Scopus (106) Google Scholar), a high precision NMR structure is available (10Sonnichsen F.D. DeLuca C.I. Davies P.L. Sykes B.D. Structure. 1996; 4: 1325-1337Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), and its ice-binding site has been defined by site-directed mutagenesis (11Jia Z. DeLuca C.I. Chao H. Davies P.L. Nature. 1996; 384: 285-288Crossref PubMed Scopus (222) Google Scholar, 15DeLuca C.I. Chao H. Sonnichsen F.D. Sykes B.D. Davies P.L. Biophys. J. 1996; 71: 2346-2355Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 16Chao H. Sonnichsen F.D. DeLuca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar, 18Baardsnes J. Davies P.L. Biochim. Biophys. Acta. 2002; 1601: 49-54Crossref PubMed Scopus (74) Google Scholar). As a result of these findings, the activities of numerous mutants and derivatives have been determined as a function of protein concentration. The intramolecular dimer was made by connecting two monomers in a tandem, head-to-tail arrangement by an RD3-type linker sequence. This construct was termed wlwAFP, for wild type-linker-wild type AFP, and its activity was compared with that of the HPLC-12 monomer. Fusions of AFPs with non-antifreeze proteins can enhance activity simply through an increase in size (26DeLuca C.I. Comley Davies P.L. Biophys. J. 1998; 74: Full Text Full Text PDF PubMed Scopus Google Scholar). this effect, one of the ice-binding sites in the dimer was by the H. Sonnichsen F.D. DeLuca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar) to a between and the dimer with one ice-binding site of the increased activity of the dimer is of an increase in the overall size of the protein and is of doubling of the area of the ice-binding to in activity from the number of ice-binding of the of these faces to a ice an was through the of a disulfide bond between C-terminal residues of an the basis of the ice-binding faces of this dimer are from with the same ice When the two AFPs were joined in tandem by the acid RD3 linker sequence their antifreeze activity was twice that of the type III monomer. However, the two AFPs were joined by the C-terminal disulfide bond their AFP activity was only than the monomer The difference between these is that the can potentially engage both ice-binding sites to the same ice in the dimer the two sites to a plane When one of the ice-binding sites of the dimer was inactivated by site-directed mutagenesis the antifreeze activity also to that of the monomer. the activity of the head-to-tail dimer is in to its increased size is of the flexible linker a doubling of the protein surface in contact with of and the for the HPLC-12 isoform of type III AFP H. Davies P.L. Sykes B.D. Sonnichsen F.D. Protein Sci. 1993; PubMed Scopus Google Scholar). Its is the of the sequence with an N-terminal for in construct was with and to the from of the to the of the antifreeze The was used to the N-terminal domain of the recombinant which a C-terminal of the C-terminal sequence is by H. Davies P.L. Sykes B.D. Sonnichsen F.D. Protein Sci. 1993; PubMed Scopus Google Scholar), was with and to the The linker sequence was from and had and sites at its and Approximately each of the and the sequence and of linker were using The were into and were on the of a a were by make wlxAFP, the same was using an of the H. Sonnichsen F.D. DeLuca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar) to the and of intramolecular were and used for the monomers H. Sonnichsen F.D. DeLuca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar) with a within the flexible linker the protein was for in an ice with the of and to both the and The protein was using with an concentration in The protein was with and The protein was in and were by acid analysis of were for thermal hysteresis in of the of was by mutagenesis PubMed Scopus Google Scholar) using the sequence as H. Davies P.L. Sykes B.D. Sonnichsen F.D. Protein Sci. 1993; PubMed Scopus Google Scholar). The protein was and from for the of to the used as the and This the protein to monomeric and prevented of the protein with the The was into at and Approximately of AFP was into in a and at for other residues are in the AFP; or intramolecular be in the disulfide-linked was from monomer by were at a of the in acid and by a of The was by in the of reducing with type III monomer and range as Protein were with of to were with to the of the disulfide Protein was by with The dimer was and in The concentration of dimer was determined by acid analysis of of the disulfide-linked dimer by was in to assess the antifreeze activity of the disulfide-linked dimer compared with monomer from the same were to with or was at The and were for thermal hysteresis and their was by and hysteresis is defined as the temperature difference between the melting point and non-equilibrium freezing point of an AFP solution. and wild type AFPs were for thermal hysteresis activity as A. Hew C.L. Eur. J. Biochem. 1991; PubMed Scopus Google Scholar). crystals were using a and ice growth of more than that the freezing point has been or Molecular of and was by the sequence to D.S. J. 1993; Google Scholar) using the RD3 NMR structure (24Miura K. Ohgiya S. Hoshino T. Nemoto N. Suetake T. Miura A. Spyracopoulos L. Kondo H. Tsuda S. J. Biol. Chem. 2001; 276: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) as a with has an acid at the C-terminal of the dimer linker and residues at the of the AFP of the This an in the However, of and a within this the structure was using to this The structure was using a and were using D.S. J. 1993; Google Scholar) The was using the by to in two type III AFP and a disulfide bond between using the the disulfide bond of the dimer was by using a which angle at the disulfide bond and the two was and were The ice-binding faces of the energetically favorable were to the of the two ice-binding faces with to one as as Type III recombinant that were in to the The dimer proteins from this from as at the same concentration as the monomer H. Davies P.L. Sykes B.D. Sonnichsen F.D. Protein Sci. 1993; PubMed Scopus Google Scholar). This is with the for and the AFP monomer being similar and that the had the hysteresis activity for was at compared with for the monomer the concentration range of to the dimer a in activity compared with the monomer. This is in with the of Wang et al. (23Wang X. DeVries A.L. Cheng C.H. Biochim. Biophys. Acta. 1995; 1247: 163-172Crossref PubMed Scopus (49) Google Scholar), the RD3 isoform at increased activity by on a molar basis compared with the RD1 and RD2 monomer This of was also reported by Miura et al. (24Miura K. Ohgiya S. Hoshino T. Nemoto N. Suetake T. Miura A. Spyracopoulos L. Kondo H. Tsuda S. J. Biol. Chem. 2001; 276: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) for recombinant RD3 at a concentration of compared with the monomer. In the the ice-binding site in the C-terminal domain has been by the This of one of the two ice-binding sites the activity of the dimer by compared with the concentration range to However, the dimer of activity were than the monomer. The increase in activity of compared with monomer is with reported by DeLuca et al. (26DeLuca C.I. Comley Davies P.L. Biophys. J. 1998; 74: Full Text Full Text PDF PubMed Scopus Google Scholar), the size of type III AFP by to other proteins increased its AFP activity proteins of III AFP with a protein and 20 III AFP with a thermal hysteresis activity of and at The wlxAFP, at this with a thermal hysteresis of at The activity of this protein between the type III monomer and protein the concentration range tested to of the dimer of from the monomer on HPLC, two of area with times of and The proteins in the first and were using as the monomer and was no of the in disulfide The of by was as with the dimer protein more to the on the area of the of the a A of the was by the of The reduction of the disulfide bond was an was with This parallel analyses of the same in both the monomer and dimer with in the protein the molar concentration of the monomer was twice that of the hysteresis activity that the dimer had more activity than the monomer at protein of and This activity is than that with the head-to-tail dimer is similar to the activity for the dimer with one ice-binding hysteresis activity of hysteresis activity of and The of the the of a structure for the wsswAFP, the of the two ice-binding sites was by AFP domains were by the disulfide bond and bonds by at each This for a of the that be by the two AFP domains the disulfide The residues of each ice-binding face are in the energetically favorable dimer structure in in The of the energetically favorable that both ice-binding faces opposite of the dimer and are of the same ice of to of recombinant dimer shows an increase in antifreeze activity as compared with the monomer. This activity increase is to the for the dimer reported by Wang et al. (23Wang X. DeVries A.L. Cheng C.H. Biochim. Biophys. Acta. 1995; 1247: 163-172Crossref PubMed Scopus (49) Google Scholar) and is within the range of determined by Miura et al. (24Miura K. Ohgiya S. Hoshino T. Nemoto N. Suetake T. Miura A. Spyracopoulos L. Kondo H. Tsuda S. J. Biol. Chem. 2001; 276: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) for this using a dimer made from identical monomers that are structurally and we show that this is a of can be using AFPs that in sequence from the AFPs in the RD3 The doubling of antifreeze activity for a protein of the same has in K.V. 1995; PubMed Scopus Google Scholar, F. of Springer-Verlag, Google Scholar) and is of We have and investigated several for this we have the effects of the size number of ice-binding sites and the area of ice-binding site in contact with of on by (26DeLuca C.I. Comley Davies P.L. Biophys. J. 1998; 74: Full Text Full Text PDF PubMed Scopus Google Scholar), there was a in activity for the recombinant dimer an domain of the domain was on the type III which was shown to be from to H. Sonnichsen F.D. DeLuca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar). the in activity be to antifreeze activity in the C-terminal This activity increase with the enhanced activity on size using N-terminal proteins (26DeLuca C.I. Comley Davies P.L. Biophys. J. 1998; 74: Full Text Full Text PDF PubMed Scopus Google Scholar). The that the in the is on the C-terminal to the The activity between the activity of the monomer and there is such a between protein and activity that it is to the of by of the from the activity of the monomer and the and it is that only a of the dimer activity is to the increased size of the than the that the enhanced activity of the type III AFP be in to two ice-binding sites the of to The dimer was to the two ice-binding sites at opposite of the The is at the of the and an disulfide bond form a dimer that is from both AFP domains engage the ice Molecular that of the energetically favorable allow of both sites to a ice The increase in activity of the dimer is with that with and can be for by the increase in size of the The dimer as a and there is no to the ice-binding site it be to is to that reduction of the disulfide bond for a between the monomer and dimer This is at AFP in concentration have a on antifreeze and are for a between AFP molecules with similar the to dimer has the same and number of ice-binding sites as the wsswAFP, is more The difference is the head-to-tail arrangement of the two domains and their flexible which the two AFPs to bind the ice surface the basis of the structure of Miura et al. (24Miura K. Ohgiya S. Hoshino T. Nemoto N. Suetake T. Miura A. Spyracopoulos L. Kondo H. Tsuda S. J. Biol. Chem. 2001; 276: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) determined that only a of the RD3 be to engage both ice-binding sites to the primary prism plane ice This is on the that only one of the NMR is to However, Antson et al. (9Antson A.A. Smith D.J. Roper D.I. Lewis S. Caves L.S. Verma C.S. Buckley S.L. Lillford P.J. Hubbard R.E. J. Mol. Biol. 2001; 305: 875-889Crossref PubMed Scopus (106) Google Scholar) have recently reported that type III AFP can bind to a of similar This includes prism planes and primary planes in to the primary prism a of ice-binding sites for RD3 than and this increase the number of active AFP in solution. The ice-binding face be to bind to the same ice plane as the for in ice-binding site of the modeled structure using that both ice-binding faces of the structure have the to engage a ice surface AFP as a of is now that antifreeze activity can be enhanced by the of the ice-binding site. The type isoform from at in has an acid compared with the isoforms and and has a ice-binding face J. Chao H. Davies P.L. Lett. 1999; PubMed Scopus Google Scholar). antifreeze twice that of the more acid isoform H. Davies P.L. Protein Sci. 1996; PubMed Scopus Google Scholar). The same is also with antifreeze from Antarctic A.L. Biochem. Physiol. A. Scopus (109) Google Scholar). These with an on the a with a of has twice the activity of and both of which have an of of antifreeze activity with ice-binding site area is the A isoform with two of is more than twice as active as the isoform Davies P.L. Jia Z. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). As the dimer has there is activity from of the AFP increase in activity the to increase contact with the ice type AFPs, and AFP are simply the protein increase the size of the ice-binding face the ice However, the type III AFPs are proteins with a defined ice-binding the surface area be increased simply by the size of the monomer. the flexible linker is an way to this As antifreeze proteins are thought to bind to the ice the ice to into a surface between The with proteins is thought to be to the protein reducing the between which in makes it more for water to join the ice (26DeLuca C.I. Comley Davies P.L. Biophys. J. 1998; 74: Full Text Full Text PDF PubMed Scopus Google Scholar). the same AFPs with ice-binding sites a on the the area between thereby reducing the of the ice on the type III RD3 isoform have shown that activity from an increase in the size of the ice-binding surface in contact with with a to the increase in the size of the acid analysis was by the of

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