Artificial Multimers of the Type III Antifreeze Protein
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m304390200
ISSN1083-351X
AutoresYoshiyuki Nishimiya, Satoru Ohgiya, Sakae Tsuda,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoA variant of antifreeze protein (AFP) named RD3 from antarctic eel pout (Lycodichthys dearborni) comprises the type III AFP intramolecular dimer, which is known to exhibit a significant enhancement of thermal hysteresis when compared with the type III AFP monomer (Miura, K., Ohgiya, S., Hoshino, T, Nemoto, N., Suetake, T., Miura, A, Spyracopoulos, L., Kondo, H., and Tsuda, S. (2001) J. Biol. Chem. 276, 1304–1310). Here we genetically synthesized intramolecular dimer, trimer, and tetramer of the type III AFP, for which we utilize the genes encoding the primary sequences of the N-domain, the C-domain, and the 9-residue linker of RD3, and we examined the AFP multimerization effects on thermal hysteresis and ice crystal morphology. Significantly, (i) the thermal hysteresis increases in proportion with the size of the multimers, (ii) a larger size of the multimer exerts the maximum activity at lower concentration, (iii) every multimer changes the morphology of a single ice crystal into a unique shape that is similar but not identical to the ordinary hexagonal bipyramid, and (iv) the size of ice crystal becomes dramatically small with increasing the concentration of the multimer. The thermal hysteresis enhancement of the multimer was detected in both molar and domain bases. These results suggest that a molecule comprising the multiple AFP domains connected in tandem acquires an enhanced affinity for the ice binding. A variant of antifreeze protein (AFP) named RD3 from antarctic eel pout (Lycodichthys dearborni) comprises the type III AFP intramolecular dimer, which is known to exhibit a significant enhancement of thermal hysteresis when compared with the type III AFP monomer (Miura, K., Ohgiya, S., Hoshino, T, Nemoto, N., Suetake, T., Miura, A, Spyracopoulos, L., Kondo, H., and Tsuda, S. (2001) J. Biol. Chem. 276, 1304–1310). Here we genetically synthesized intramolecular dimer, trimer, and tetramer of the type III AFP, for which we utilize the genes encoding the primary sequences of the N-domain, the C-domain, and the 9-residue linker of RD3, and we examined the AFP multimerization effects on thermal hysteresis and ice crystal morphology. Significantly, (i) the thermal hysteresis increases in proportion with the size of the multimers, (ii) a larger size of the multimer exerts the maximum activity at lower concentration, (iii) every multimer changes the morphology of a single ice crystal into a unique shape that is similar but not identical to the ordinary hexagonal bipyramid, and (iv) the size of ice crystal becomes dramatically small with increasing the concentration of the multimer. The thermal hysteresis enhancement of the multimer was detected in both molar and domain bases. These results suggest that a molecule comprising the multiple AFP domains connected in tandem acquires an enhanced affinity for the ice binding. Antifreeze protein (AFP) 1The abbreviations used are: AFP, antifreeze protein; RD3, native type III AFP intramolecular dimer comprising the N- and C-domains connected in tandem through a 9-residue linker sequence; RD3Nl, the N-domain of RD3 with the linker; RD3NN, the intramolecular dimer comprising the two N-domains connected in tandem by the linker; RD3NNC (RD3NCN, RD3NCC), the intramolecular trimer comprising the N- and C-domains connected in tandem by the linker; RD3NCNC, the intramolecular tetramer comprising the two RD3 molecules connected in tandem by the linker; T f, freezing temperature; T m, melting temperature; TH, thermal hysteresis.1The abbreviations used are: AFP, antifreeze protein; RD3, native type III AFP intramolecular dimer comprising the N- and C-domains connected in tandem through a 9-residue linker sequence; RD3Nl, the N-domain of RD3 with the linker; RD3NN, the intramolecular dimer comprising the two N-domains connected in tandem by the linker; RD3NNC (RD3NCN, RD3NCC), the intramolecular trimer comprising the N- and C-domains connected in tandem by the linker; RD3NCNC, the intramolecular tetramer comprising the two RD3 molecules connected in tandem by the linker; T f, freezing temperature; T m, melting temperature; TH, thermal hysteresis. has been identified from bacteria to animals, which is presumably a product of their cold adaptation processes to defend their tissues from freezing injury (1Fletcher G.L. Hew C.L. Davies P.L. Annu. Rev. Physiol. 2001; 63: 359-390Crossref PubMed Scopus (398) Google Scholar). The water solution of AFP exhibits a non-colligative freezing temperature (T f) depression, which is 300–500-fold effective compared with the equilibrium colligative depression observed for the ordinary solutes (on a molar basis) (2Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-618Crossref PubMed Scopus (467) Google Scholar). To date, it appeared that such a splendid T f depression of AFP is a result of its ice binding ability, which inhibits the growth of embryo ice crystals that naturally emerged in the supercooling water. This ice growth inhibitory function of AFP does not change significantly the melting temperature (T m) of the solution so that it gives a difference between T f and T m, which is generally termed thermal hysteresis (TH) (2Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-618Crossref PubMed Scopus (467) Google Scholar). Because such a unique inhibitory function of AFP exhibits a high potential for the cryo-industrial usages, such as cryo-preservations of tissues and cells and maintaining the texture of frozen materials (3Carpenter J.F. Hansen T.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8953-8957Crossref PubMed Scopus (175) Google Scholar, 4Chao H. Davies P.L. Carpenter J.F. J. Exp. Biol. 1996; 199: 2071-2076PubMed Google Scholar), it becomes more and more significant to design and produce the activity-improved variants of AFP based on the detailed understanding of its ice-binding mechanism. An elegant explanation for the non-colligative depression of T f of AFP was made by "adsorption-inhibition mechanism," in which accumulations of AFPs onto the growing surfaces of ice crystal result in the creations of numbers of convex ice surfaces at the limited open spaces on the ice between the bound AFPs (5Raymond J.A. DeVries A.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2589-2593Crossref PubMed Scopus (675) Google Scholar). The free energy of the convex ice surfaces becomes larger with the increase of the surface curvature (so-called Kelvin effect (6Knight C.A. Cheng C.C. DeVries A.L. Biophys. J. 1991; 59: 409-418Abstract Full Text PDF PubMed Scopus (462) Google Scholar)). Therefore, the further binding of water molecules onto the convex ice surfaces becomes energetically unfavorable, leading to the suppression of the ice crystal growth by increasing the number of bound AFPs. This irreversible ice binding model was then modified to a semi-reversible model (7Hew C.L. Yang D.S.C. Eur. J. Biochem. 1992; 203: 33-42Crossref PubMed Scopus (172) Google Scholar, 8Wen D. Laursen R.A. Biophys. J. 1992; 63: 1659-1662Abstract Full Text PDF PubMed Scopus (145) Google Scholar) implicated in the kinetics of AFP-induced ice growth inhibition, which includes the following AFP adsorption steps (9Chapsky L. Rubinsky B. FEBS Lett. 1997; 412: 241-244Crossref PubMed Scopus (50) Google Scholar): (i) attachment to the ice-water interface; (ii) rearrangement of adsorbed molecules by diffusion, reorientation, and/or conformational change; and (iii) detachment from the interface. It was assumed that such progressive steps of AFP adsorption are highly correlated with the local concentration of AFPs around the ice crystal surface (2Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-618Crossref PubMed Scopus (467) Google Scholar). Here we focused a 7-kDa small globular type III AFP (10Li X.-M. Trinh K.-Y. Hew C.L. Buettner B. Baenziger J. Davies P.L. J. Biol. Chem. 1985; 260: 12904-12909Abstract Full Text PDF PubMed Google Scholar) and its genetically synthesized dimer, trimer, and tetramer to investigate the AFP multimerization effects on the T f depression and the ice crystal morphology. The type III AFP has been studied extensively for the species identified from Macrozoarces americanus (Atlantic ocean pout) and appeared to possess a diversity in amino acid sequences by multiple gene encodings (10Li X.-M. Trinh K.-Y. Hew C.L. Buettner B. Baenziger J. Davies P.L. J. Biol. Chem. 1985; 260: 12904-12909Abstract Full Text PDF PubMed Google Scholar, 11Hew C.L. Wang N.-C. Joshi S. Fletcher G.L. Scott G.K. Hayes P.H. Buettner B. Davies P.L. J. Biol. Chem. 1988; 263: 12049-12055Abstract Full Text PDF PubMed Google Scholar). Determinations of the three-dimensional x-ray structures (12Jia Z. DeLuca C.I. Chao H. Davies P.L. Nature. 1996; 384: 285-288Crossref PubMed Scopus (222) Google Scholar, 13Yang D.S.C. 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) and the high precision NMR structure (14Sönnichsen F.D. DeLuca C.I. Davies P.L. Sykes B.D. Structure. 1996; 15: 1325-1337Abstract Full Text Full Text PDF Scopus (165) Google Scholar) revealed that type III AFP constructs a globular shape characterized by "2-fold symmetric motif" or a "pretzel fold." It also revealed that the putative ice-binding residues (e.g. Gln9, Asn14, Thr15, Thr18, and Gln44), which were identified by observing the effect of site-directed mutations of these residues on the TH activity, are located in the planar surface of the type III AFP with tight atom packing, so as to undergo a specific interaction with the ice prism plane (15DeLuca C.I. Davies P.L. Ye Q. Zia Z. J. Mol. Biol. 1998; 275: 515-525Crossref PubMed Scopus (80) Google Scholar). In 1995, RD3 was identified as an exceptional isomer of the type III AFP from Lycodichthys dearborni (Antarctic ocean pout) and appeared to comprise the two type III AFP domains connected in tandem through a 9-residue linker sequence (-Asp-Gly-Thr-Thr-Ser-Pro-Gly-Leu-Lys-) (16Wang X. DeVries A.L. Cheng C.H. Biochim. Biophys. Acta. 1995; 1247: 163-172Crossref PubMed Scopus (49) Google Scholar). The three-dimensional structure of RD3 was determined using multidimensional NMR techniques (17Miura 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), which revealed the following: (i) the N- and C-domains construct the ordinary type III AFP structures that are laterally oriented with an angle of 32 ± 12°, and (ii) a flexible bend of the 9-residue linker sequence brings the two ice-binding planes of RD3 to an allowed range of orientation. Interestingly, it appeared that TH activity of RD3 is not a simple additive activity of the two type III AFP monomers but exhibits about 6-fold enhancements compared with the monomer (on a molar basis) in the lower concentration range (0–0.5 mm) (17Miura 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). This significant TH enhancement of RD3 was ascribed to some cooperative effect originating from the two proximal domains; however, no further information has been obtained for considering detailed tandem repetition effects of the AFP domains on the thermal hysteresis and the ice crystal morphology. In the present study, we attempted to design and generate new intramolecular dimer, trimer, and tetramer of the type III AFP, for which we utilized the primary sequences of either N- or C-domain of RD3. For example, RD3NNC denotes a protein comprising the tandem connections of the two successive N-domain sequences plus the C-domain sequence of RD3. We succeeded in generating RD3NN, RD3NNC, RD3NCN, RD3NCC, and RD3NCNC, for which connections of the AFP domains utilize the 9-residue linker sequence of the native RD3. For these artificial AFP multimers, we measured and compared the TH activity and the ice crystal morphology. Significantly, these AFP multimers exhibit different levels of enhanced TH activity compared with the type III AFP monomer at a lower AFP concentration range on molar and domain bases. It further appeared that these AFP multimers affect the ice crystal morphology in different ways. We believe that these results will significantly expand our understandings of the ice-binding mechanism of AFP and its effect on the TH activity and the ice crystal morphology, which will lead to the design and the production of an activity-modified or -enhanced variant of AFP. Construction of DNAs Encoding the Type III AFP Multimers—Our previously synthesized genes of RD3 (17Miura 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) and RD3Nl (18Miura K. Ohgiya S. Hoshino T. Nemoto N. Odaira M. Nitta K. Tsuda S. J. Biochem. (Tokyo). 1999; 126: 387-394Crossref PubMed Scopus (11) Google Scholar) were utilized for the present constructions of DNAs to express the type III AFP intramolecular dimer, trimer, and tetramer. A gene encoding RD3 was newly prepared, in which we removed KpnI and SmaI restriction sites located in a portion encoding the 9-residue linker by using the site-directed mutagenesis (denoted as RD3ΔSma/Kpn). By using this as a template, we further prepared a gene that encodes 6–9 residues of the linker plus RD3 (denoted Linker-RD3) by PCR using a forward primer (5′-CGCGGATCCTATTCGTAGTTTTTAACCATG-3′) and a backward primer (5′-GGAACTGCAGCCCGGGTCTGAAATCCGTTGTTGCTAACCAG-3′). It is noted that the gene encoding the part of the linker is designed so as to include SmaI restriction site. Likewise, a gene encoding RD3 plus linker (RD3-Linker) was constructed using a forward primer (5′-GTCCCCCGGGGAGGTGGTGCCGTCTTCGTAGTTTTTAACCATGTCCGGCATCAGGGTCTG-3′) and a backward primer (5′-GAGCTGCAGTTAACTTTAAG-3′). In this case, the gene encoding 1st to 6th residues of the linker was prepared to include only SmaI restriction site. In addition, the gene encoding a part of the linker (including the SmaI restriction site) connected with that encoding the N-domain of RD3 (denoted as Linker-RD3N) was amplified using a forward (5′-CGCGGATCCTATTCGTAGTTTTTAACCATG-3′) and a backward primer (5′-GGAACTGCAGCCCGGGTCTGAAATCCGTTGTTGCTAACCAG-3′) by using the gene encoding the N-domain of RD3 as a template. Combined use of these prepared DNA fragments (RD3ΔSma/Kpn, Linker-RD3, Linker-RD3N, RD3-Linker, RD3, and RD3Nl) made it possible to construct the genes encoding RD3NN, RD3NCC, RD3NNC, RD3NCN, and RD3NCNC as summarized in Fig. 1, for which the DNA sequences were occasionally digested with NdeI and BamHI followed by ligations with a vector pET20b (Novagen). Expressions and Purifications of the Recombinant Multimers—The plasmid DNAs were transformed into Escherichia coli strain BL 21 (DE3), which was grown at 28 °C in LB medium supplemented with 100 μg/ml ampicillin until the cell growth reaches to the early stationary phase. To induce the expression of any recombinant AFP multimer, 0.5 mm isopropyl-d-thiogalactopyranoside was added, and the cultures were grown at 28 °C for overnight. The purification of the multimers was commonly performed according to the methods described previously (19Chao H. Sönnichsen F.D. DeLuca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar). The culture was centrifuged at 4,000 × g for 30 min at 4 °C, and then we sonicated the precipitated cell pellet containing the inclusion body of the AFP multimers. After the sonication, the fraction containing the AFP multimers was collected by centrifugation at 12,000 × g for 30 min at 4 °C, and then it was washed with 0.1% (v/v) Triton X-100, 1 mm EDTA for three times, and dissolved into 100 mm Tris-HCl (pH 8.5) containing 6 m guanidine hydrochloride at room temperature. Each dissolved AFP multimer was diluted with 50 mm K2HPO4 containing 100 mm NaCl (pH 10.7) at 4 °C followed by extensive dialysis against 50 mm sodium acetate (pH 3.7) at 4 °C. The precipitant formed during dialysis was removed occasionally by centrifugation. The supernatant containing AFP was loaded onto a high-S column (Bio-Rad), and the column-bound AFP was eluted with a linear NaCl gradient (0–0.5 m) using 50 mm sodium acetate buffer (pH 3.7). The fractions containing the isolated AFP were stored and dialyzed against 0.1 m ammonium bicarbonate (pH 7.9). The purity and the molecular weights (14 (dimer), 22 (trimers), and 29 kDa (tetramer)) of the samples were checked by 16% SDS-PAGE. Measurements of Thermal Hysteresis and Ice Crystal Morphology— The T f measurements were performed for the recombinant AFP multimers dissolved in 0.1 m ammonium bicarbonate (pH 7.9) by using an osmometer (model OM 802, Vogel). The observation of ice crystal morphology and the T m determination were performed using an in-house photomicroscope system consisting of Leica DMLB 100 photomicroscope equipped with a Linkam LK600 (liquid nitrogen-type) temperature controller and a CCD camera. The measurements of T f and T m were repeated three times using the fresh samples, and the averaged values were used for determination of the TH activity (TH = T f – T m). The TH values of the multimers were examined as a function of the protein concentration ranging from 0.025 to 0.6 mm. For the observation of ice crystal morphology, a droplet (∼1.0 μl) of the sample solution was once frozen and subsequently heated until a single ice crystal was observed separately in the solution by manipulation of the temperature controller. The morphological change of the single ice crystal into a hexagonal bipyramid caused by the accumulations of AFP on the ice surfaces was then observed with the cooling rate of 0.01 °C per min. The ice crystal morphology observed under different temperatures, cooling rates, and elapses of time were recorded as a movie file on a personal computer. The sample volume and the cooling rate were kept constant through the measurements. We successfully prepared the samples of recombinant type III AFP multimers, such as RD3Nl as the monomer, RD3 and RD3NN as the dimers, RD3NCC, RD3NNC, and RD3NCN as the trimers, and RD3NCNC as the tetramer (Fig. 1), which were used for the measurements of TH activity and the observation of ice crystal morphology. Fig. 2a plots the TH activity (ΔT f (°C)) of the AFP multimers in the molar concentration range between 0 and 0.6 mm. As can be seen, the activity of the monomer (RD3Nl) is dramatically improved in the dimers (RD3 and RD3NN), and the activities of the dimers are further improved in the trimers (RD3NNC, RD3NCC, and RD3NCN). No significant difference in the TH activity was detected between the trimers and a tetramer (RD3NCNC), although the tetramer exhibits the highest activity. As a consequence, the activity curves of Fig. 2a were largely divided into three groups corresponding to monomer, dimers, and the others. Interestingly, it appeared that the TH activity curves of the multimers are sigmoid in shape, which might be ascribed to a kinetic icebinding process of the multimers. The differences between the activity curves of Fig. 2a are distinctly identified in Fig. 2b, which plots the TH activity ratio between ΔT f (multimers) and ΔT f (monomer). For the two recombinant dimers, it appeared that RD3NN is less active than RD3 in the concentration range between 0.02 and 0.2 mm. The maximum ratios of RD3 and RD3NN were estimated to be 8.4 and 6.5, respectively. Among the three trimers, RD3NNC and RD3NCC possessed almost identical activity through the whole concentration range, whereas RD3NCN did not catch up to the activity levels of the other two in the concentration range between 0.02 and 0.15 mm. The maximum ratios of RD3NNC, RD3NCC, and RD3NCN were estimated to be 22.6, 22.0, and 16.8, respectively. A small enhancement of the relative activity was distinctly detected between RD3NCNC and the others for a small range of AFP concentrations between 0.02 and 0.1 mm. The maximum ratio of RD3NCNC was estimated to be 24.6. Overall, the ΔT f (multimers)/ΔT f (monomer) ratio increases in keeping with the number of domain multiplications, although the two species that comprise the N-domain at the C-terminal end (i.e. RD3NN and RD3NCN) tends to lower the activity. Another significant observation of Fig. 2b is that the top position of each curve shifts to the lower concentration side with increasing the size of the multimers, suggesting that a larger size of the multimer exerts the maximum activity at a lower AFP concentration. It is noted that we have reported the detection of maximum 5.9-fold activity enhancements of RD3 compared with the monomer (17Miura 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). It appeared that our previous estimation is slightly insufficient presumably due to an imperfect dissolution of the protein before the TH measurements, and should be revised to maximum 8.4-fold enhancements in the present study. Fig. 2c shows the TH activity of the multimer plotted as a function of the "domain concentration," in which the ΔT f values of the dimer, trimer, and tetramer are plotted against the molar concentration (mm) times 2, 3, and 4, respectively. This implies that the longitudinal axis of Fig. 2c represents a "per domain ΔT f," so that the observed difference between the curves of Fig. 2c reflects the tandem repetition-induced change of the TH activity of an AFP domain comprised in a certain multimer. As can be seen in Fig. 2c, the per domain ΔT f values of every multimer are notably improved when compared with that of the monomer (RD3Nl). For dimers, it detected a difference in the profile between RD3 and RD3NN; the activity of the former is highly improved compared with the latter. For trimers, the highest and similar profiles of the per domain ΔT f curves are obtained for RD3NCC and RD3NNC, whereas less improvement is detected for RD3NCN. An interesting finding is that the activity profile of a tetramer RD3NCNC is almost identical to that of RD3, which implies that the TH activity of the tetramer is not significantly enhanced at a per domain level. It should be noted that in Fig. 2c the curves of the multimers are sigmoid in shape, and the species that comprise the N-domain at the C-terminal end tend to lower the activity as already described. It was indicated that both two ice-binding planes constructed in RD3 are able to interact with the prismic planes of a single hexagonal ice crystal, which results in the morphological change of the ice crystal into a unique bipyramidal shape (17Miura 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). In the present study, the ice crystal morphology was examined for each water solution of the multimer under two different protein concentrations (0.003 and 0.05 mm). As shown in the photomicrographs of Fig. 3, every multimer changes the morphology of a single ice crystal into a diamond shape, which is closely similar to the ordinary hexagonal bipyramid observed for RD3 and various AFPs. Differently from the ordinary bipyramid, the ice crystals created by the multimers commonly lack the acute corners around the pyramidal junction that slightly protrudes to the a axis direction. It is also noted that the pyramidal plane acquires a slight curvature. As for RD3Nl, it does not create a bipyramidal ice crystal at the concentration of 0.003 mm, and it creates an incomplete bipyramid without acute tips when its concentration reaches 0.02 mm (Fig. 3). It is interesting to note that the c/a axial ratio is about 2 for RD3NN at the protein concentration of 0.003 mm, which slightly decreases in proportion with the size of the multimer, and is estimated to be 1.5 for RD3NCNC. Another significant observation is that the ice crystal size becomes dramatically small (1/3 to 1/4) only in the case of multimers by increasing their protein concentration from 0.003 to 0.05 mm. All of these data imply that a molecule comprising the multiple AFP domains connected in tandem acquires a unique manner of the ice binding. Here we examined the tandem repetition effects of the type III AFP domains on TH activity and ice crystal morphology, and we identified for the first time the following. (i) The TH activity increases in proportion with the size of the multimers. (ii) A larger size of the multimer exerts the maximum activity at lower concentrations. (iii) Every multimer changes the morphology of a single ice crystal into a unique shape that is similar but not identical to the ordinary hexagonal bipyramid. (iv) The size of ice crystal becomes dramatically small by increasing the concentration of the multimer. Interestingly, the TH activity curves against the protein concentrations are sigmoid in shape (Fig. 2), which is presumably a consequence of the kinetic ice binding process of each AFP multimer. Wen and Laursen (8Wen D. Laursen R.A. Biophys. J. 1992; 63: 1659-1662Abstract Full Text PDF PubMed Scopus (145) Google Scholar) proposed a two-step ice-binding process of AFP, in which they assumed an existence of a reversible hydrogen-bonding mechanism between AFP and ice surface at a low range of AFP concentration. Once the AFP concentration becomes high, the AFP molecules on the ice surface begin to pack and interact with each other so as to increase a total affinity of the ice binding (2Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-618Crossref PubMed Scopus (467) Google Scholar, 8Wen D. Laursen R.A. Biophys. J. 1992; 63: 1659-1662Abstract Full Text PDF PubMed Scopus (145) Google Scholar). Chao et al. (20Chao H. DeLuca C.I. Davies P.L. FEBS Lett. 1995; 357: 183-186Crossref PubMed Scopus (49) Google Scholar) and DeLuca et al. (21DeLuca C.I. Comley R. Davies P.L. Biophys. J. 1998; 74: 1502-1508Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), however, reported that AFP molecules are independently active, and the protein-protein interaction is not an indispensable requirement for the tight binding to ice. For the present AFP multimers, on-rates of the consecutive ice bindings of the comprised domains should be significantly influenced by each other, since they are in close proximity by the connections through the linker sequences. Hence, the observations of the sigmoid curves of Fig. 2 would be attributed to "intra"-molecular cooperative interactions between the AFP domains of a multimer. It could be further assumed that the cooperative interactions are presumably strengthened in keeping with the number of domain multiplications and with the protein concentration as well. The TH activity enhancement therefore occurs at both molar and domain bases (Fig. 2), and the size of an ice crystal dramatically decreases by increasing the protein concentration of the multimer (Fig. 3). An interesting observation is that the largest enhancement of the TH activity was observed for the trimers but not for a tetramer; the maximum activity ratios of the two trimers (22.6 (RD3NNC) and 22.0 (RD3NCC)) are about 3-fold higher than that of a natural dimer (8.4 (RD3)) (Fig. 2b). In order to consider the present detected domain multiplication effects on the TH activity and ice crystal morphology from the three-dimensional structural viewpoint, we constructed model structures of the trimers and a tetramer based on our previous determinations of the NMR structure of RD3 (PDB code 1C8A) (17Miura 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). Fig. 4 illustrates one of the model structures for RD3NCNC, in which (i) two molecules of RD3 are connected using the coordinates of the native RD3 linker sequence and (ii) the N-terminal three residues (-Asn-Lys-Ala-) are deleted from the second NC molecule, because these three residues do not exist in the second domain of native RD3 (16Wang X. DeVries A.L. Cheng C.H. Biochim. Biophys. Acta. 1995; 1247: 163-172Crossref PubMed Scopus (49) Google Scholar, 17Miura 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). The previous study demonstrated that the N- and C-domains of RD3 are laterally oriented with an angle of 32 ± 12°, and the ice binding plane of the N-domain is located ∼3.5 Å behind that of the C-domain in an averaged structure (17Miura 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). Furthermore, it demonstrated that the linker portion is flexible and has an ability to bring the two ice-binding planes of RD3 to an allowed range of orientation; the two planes are presumably aligned with respect to the ice surface in the ice-bound form (17Miura 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). These structural features are preserved in the model structure of RD3NCNC, leading to a twisted formation of the ice binding planes (indicated by ellipses in Fig. 4). For clarity, we rename this tetramer RD3N1C2N3C4 so as to indicate the ordinal numbers of the comprised domains. Suppose that this tetramer undertakes the manner of ice binding previously assumed for RD3 (17Miura 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), the C4-domain of RD3N1C2N3C4 located in front presumably binds first to the ice prism plane in the [0001] direction. The N3-domain may cause the second ice binding to the same prism plane in the [01¯11] direction, which is predicted by (i) position matches of the hydrophilic (15DeLuca C.I. Davies P.L. Ye Q. Zia Z. J. Mol. Biol. 1998; 275: 515-525Crossref PubMed Scopus (80) Google Scholar) and hydrophobic (22Baardsnes J. Davies P.L. Biochim. Biophys. Acta. 2002; 1601: 49-54Crossref PubMed Scopus (74) Google Scholar) atoms constructing the putative ice binding planes of the N3-domain with the spacing of water atoms of the ice prism plane and (ii) a 32° difference in the lateral alignment of the ice binding planes of the N3- and C4-domains (Ih: c axis = 7.361 Å; a axis = 4.507 Å; Ref. 23Hobbs P.V. Ice Physics. Oxford University Press, London1974: 18-39Google Scholar). Similarly, the 3rd ice binding of the C2-domain of RD3N1C2N3C4 is assumed against the same prism plane in the [21¯1¯1] direction, which is predicted by a total of ∼64° lateral alignment of the C2-domain against the C4-domain. The 4th ice binding of the N1-domain of RD3N1C2N3C4 might be further assumed against the same prism plane in the [21¯1¯0] direction. Obviously, it not possible to predict accurately the target ice surfaces of RD3NCNC especially for its third and fourth ice bindings, because it is extremely difficult to know the atom positions of putative ice binding residues in the AFP domains connected through the flexible linkers and their position matches with the water atom spacing of the ice {1011¯0} prism plane. Nevertheless, it is presumed that any domains of RD3NCNC possess the ability for specific ice binding to strengthen the interaction between the protein and ice, which is thought to be a key determinant to enhance the TH activity at molar and domain bases. Such an enhancement of the ice binding ability of the multimer is also identified by the change of the ice crystal morphology; the c/a-axial ratio becomes smaller by increasing the size of the multimer (Fig. 3). DeLuca et al. (21DeLuca C.I. Comley R. Davies P.L. Biophys. J. 1998; 74: 1502-1508Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) reported that genetically expressed type III AFP connected with thioredoxin (12 kDa) or maltose-binding protein (42 kDa) possessed only 2–3-fold higher TH activity than the type III AFP monomer. This small enhancement was ascribed to just a wider coverage of the ice surface pursued by the connected proteins that exhibit no antifreeze activity by themselves. The fact that the tetramer only slightly increases the molar basis TH activity (Fig. 2b) therefore suggests that there are significant imperfections of the ice binding between the fourth AFP domain and the ice crystal surface. One may attribute such imperfections to the structural and dynamic natures of the linker but not to an inherent ice binding ability of the fourth AFP domain. A notable observation is that the multimers containing the N-domain sequence at the C terminus (i.e. RD3NN and RD3NCN) significantly lower their TH activities (Fig. 2, b and c). One of the plausible explanations for this result is that the strength of ice binding is different between the N- and C-domains presumably due to their 20% sequence difference (16Wang X. DeVries A.L. Cheng C.H. Biochim. Biophys. Acta. 1995; 1247: 163-172Crossref PubMed Scopus (49) Google Scholar), although both of them construct a highly similar type III AFP structure (17Miura 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) comprising the same numbers of the putative ice binding residues (Gln9, Asn14, Thr15, Thr18, and Gln44 for the N-domain and Gln79, Asn84, Thr85, Thr88, and Gln144 for the C-domain) (15DeLuca C.I. Davies P.L. Ye Q. Zia Z. J. Mol. Biol. 1998; 275: 515-525Crossref PubMed Scopus (80) Google Scholar). It should be noted that the averaged order parameter (S2) of the backbone dynamics of the N-domain is slightly smaller than that of the C-domain (17Miura 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). To summarize, we succeeded to synthesize intramolecular dimer, trimer, and tetramer of the type III AFP, and we examined their antifreeze activities. They exhibit a significant enhancement of thermal hysteresis on molar and domain bases and also an enhanced ability to modify the morphology and size of a single ice crystal. The present concept to design an activityenhanced variant of AFP by tandem repetitions of AFP domains is potentially applicable to any type of AFP (i.e. types I–IV and β-helical AFPs). Such approaches might realize a production of an artificial variant of AFP that exerts an extraordinary function. We thank Dr. Hidemasa Kondo for producing the coordinate of the multimers and Ryoko Satou for technical assistance. Download .pdf (.15 MB) Help with pdf files
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