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Báo cáo khoa học: Effect of annealing time of an ice crystal on the activity of type III antifreeze protein pdf

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Effect of annealing time of an ice crystal on the activity of type III antifreeze protein Manabu Takamichi 1,2 , Yoshiyuki Nishimiya 1 , Ai Miura 1 and Sakae Tsuda 1,2 1 Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira, Sapporo, Japan 2 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita, Sapporo, Japan Antifreeze proteins (AFPs) are a structurally diverse class of macromolecules that interact with water mole- cules located in the surface of an ice crystal at temper- atures below the melting point of the solution [1]. Interaction of a substantial number of AFPs with the ice surface modifies the shape of the ice crystal, result- ing in unique morphologies, such as a hexagonal bipyramid or hexagonal trapezohedron [2]. AFPs inhi- bit ice growth by adsorbing onto the ice surface (adsorption–inhibition model) [3,4] as the temperature is lowered, and such inhibition becomes insufficient below a certain temperature that is favourable for the initiation of crystal growth. For AFP solutions, this ‘ice-growth initiation temperature’ (T ini ) is different from the melting point (T m ) of the ice crystal, and the T ini is referred to as the nonequilibrium freezing point (nonequilibrium T f ). The difference between T m and the nonequilibrium T f is defined as thermal hysteresis (TH), and is generally used as a measure of the growth inhibition ability of an AFP [5]. Therefore, determina- tion of the T ini and T m of a seed ice crystal is an essen- tial procedure to evaluate the TH activity of AFP. However, the mechanism of ice binding that alters the TH value remains unclear [6]. The TH value of AFPs has been evaluated using a nanolitre osmometer and the ‘capillary technique’ [5,7]. In the former, submicrolitre volumes of an AFP solution are introduced into an oil droplet, the temper- ature of which is controlled by a Peltier device. The TH value is determined by observing the growth of an Keywords adsorption–inhibition; annealing time; notched-fin eelpout; thermal hysteresis; type III antifreeze protein Correspondence S. Tsuda, Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Sapporo 062-8517, Japan Fax: +81 11 857 8983 Tel: +81 11 857 8912 E-mail: s.tsuda@aist.go.jp Website: http://unit.aist.go.jp/rigb/fprg/ index_e.html (Received 26 July 2007, revised 29 Septem- ber 2007, accepted 24 October 2007) doi:10.1111/j.1742-4658.2007.06164.x Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evalu- ated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8). TH of nfeAFP8 dramat- ically decreased when using a fast cooling rate (0.20 °CÆmin )1 ). It also decreased with increasing seed crystal size under a slow cooling rate (0.01 °CÆmin )1 ), but such dependence was not detected under the fast cool- ing rate. TH was enhanced 1.4- and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25 °C below T m , respectively. After anneal- ing for 2 h at 0.25 °C below T m , TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2–3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption–inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2–3 h time scale, which results in the TH dependence on the annealing time. Abbreviations AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP8, an isoform of type III AFP from Notched-fin eelpout; TH, thermal hysteresis. FEBS Journal 274 (2007) 6469–6476 ª 2007 The Authors Journal compilation ª 2007 FEBS 6469 ice crystal on the device through a microscope within a cooling gradient. Previous studies have used a similar procedure for the capillary technique with a larger ice crystal [5,6]. Duman (2001) demonstrated that consid- erably higher levels of TH are determined using the nanolitre osmometer compared with the capillary tech- nique for the same AFP sample [6]. The sample used was the AFP of the beetle Dendroides canadensis, which displayed TH of 1.4 °C using the capillary tech- nique, and 5.5 °C using the nanolitre osmometer [6]. In addition, several physicochemical factors influence TH activity [8], and there is a linear and negative cor- relation between TH activity and the logarithm of the mass fraction of ice in a sample for cerambycid beetle Rhagium inquisitor AFP [9,10]. A similar observation was also reported for the common mealworm Tenebrio molitor, for which differential scanning calorimetry was employed [11]. For fish AFPs, TH activity of approximately 1 °Cis close to the maximum value observed. Fish AFPs can be classified into four types (I–IV) or as antifreeze gly- coproteins (AFGPs). Type I AFP, with relative mole- cular weights ranging from 3.3 to 5 kDa, contains alanine-rich amphipathic a-helices [12]. Type II AFP is a globular protein with an approximate relative molecu- lar weight of 14 kDa that exhibits high structural homology to a carbohydrate-recognition domain of C-type lectin [13]. Type III AFP is a 6.5 kDa compact globular protein characterized by a unique internal two- fold symmetry motif [14]. Type IV is assumed to form a four-helix bundle structure [15]. AFGPs are glycopro- teins whose relative molecular weight ranges from 3 to 34 kDa, and comprises a repetitive tripeptide (Ala-Ala- Thr) whose Thr side chain is modified by a disaccharide moiety [16]. The structural characteristics of these fish AFPs and their target ice crystal surface are different from those of insect AFPs. A faster cooling rate decreases the apparent TH value of fish AFPs, and lar- ger ice crystals tend to initiate crystal growth at higher temperatures [17]. The TH activity of a small species of AFGP depends on the freezing rate (i.e. cooling bath temperature), while a large AFGP showed no such dependence [18]. Chapsky and Rubinsky (1997) exam- ined the kinetic ice binding of fish type I AFP using a technique called temperature-gradient thermometry [19]. For fish type III AFP, no time-dependence of TH activity has been examined to date. These data raise the question about the key determinant that has a signifi- cant influence on the TH activity of an AFP species. The present study examined the TH value of a recombinant 65-residue type III AFP called nfeAFP8, which was recently discovered in the Notched-fin eelpout [20]. nfeAFP8 exhibits 94% sequence identity with HPLC12, a well-examined type III AFP isoform found in the ocean pout Macrozoarces americanus [21]. For TH measurements, we utilized a custom-made photomicroscope system equipped with a temperature controller and a sample holder, for which data consis- tency with a commercial freezing-point osmometer had been verified. We then examined the dependence of TH value on the cooling rate, the seed ice crystal size, crystal annealing time, and annealing temperature. The data obtained revealed that the TH value is signifi- cantly increased when the ice crystal is annealed for a long time at a lower annealing temperature. Based on these results, we discuss the ice-binding mechanism of type III AFP that causes TH enhancement over a very slow time scale. Results Before determining the TH activity of nfeAFP8, the performance of our photosystem was verified using solutions of NaCl and glucose. The sample solution in the capillary tube (Fig. 1) was initially cooled with the temperature controller until completely frozen. The sample was then warmed until an ice crystal was apparent. As expected, the ice crystal prepared in each sample exhibited a ‘disk-like’ morphology, which did not subsequently change [22]. We carefully manipu- lated the temperature controller to fix the diameter of the seed crystal at approximately 20 lm, and examined whether there was a difference between T ini and T m . It appeared that the temperatures were affected by a change in the temperature controller of only ± 0.01 °C, consistent with equality between T ini and T m for the solutions of NaCl and glucose, affirming that they possess no specific ice-binding ability (i.e. T ini ¼ equilibrium T f ). In Fig. 2A, the concentration dependence of the equilibrium T f (¼ T m ) for NaCl and glucose are shown by open circles and open squares, respectively. The corresponding data measured using a freezing-point osmometer are also plotted in Fig. 2A (closed circles and closed squares). The linear profiles obtained with our photosystem and the osmometer perfectly overlapped for the NaCl and glucose solu- tions. These results indicated that our system precisely evaluates the T ini and T m values of a seed ice crystal in a solution. An example of TH determination for nfeAFP8 using our photosystem is shown in Fig. 2B, for which nfeAFP8 was dissolved to a final concentration of 0.1 mm. The figure depicts a series of images of an ice crystal formed in solution at )0.46 °C; the crystals had a bipyramidal structure due to adsorption of nfeAFP8 onto their surfaces. Adsorption of the AFP stopped ice Time-dependent enhancement of thermal hysteresis M. Takamichi et al. 6470 FEBS Journal 274 (2007) 6469–6476 ª 2007 The Authors Journal compilation ª 2007 FEBS crystal growth during the 0.01 °CÆmin )1 temperature decrease (images a and b), but failed to stop growth at )0.81 °C (image c) and allowed subsequent crystal growth (images d–h). The nonequilibrium T f of nfeAFP8 was hence determined to be )0.81 °C. As a separate experiment determined the T m to be )0.30 °C for an ice crystal in the same sample, 0.51 °C is the TH value of 0.1 mm nfeAFP8 under these experimen- tal conditions. Figure 3A shows the concentration dependence of the TH activity of nfeAFP8 measured at cooling rates of 0.01 °CÆmin )1 (closed circles) and 0.20 °CÆmin )1 (open circles). The TH activity obtained with a slow cooling rate (0.01 °CÆmin )1 ) was significantly (1.7-fold) higher than that obtained with the fast cooling rate (0.20 °CÆmin )1 ). Figure 3B is a plot of the TH depen- dence on the cooling rate of 0.1 mm nfeAFP8. The TH value decayed exponentially with increasing cooling rate, and reached a plateau at a rate of approximately 0.10 °CÆmin )1 . When the relationship between TH and crystal size was assessed, a marked difference was noted between the cooling rates of 0.01 and 0.20 °CÆmin )1 (Fig. 3C). The TH activity for a slow rate of cooling (0.01 °CÆmin )1 ) markedly decreased with increasing ice crystal size, while that measured using a fast cooling rate (0.20 °CÆmin )1 ) exhibited only a slight decrease. A high TH value (approximately 0.7 °C) was obtained only when a slow cooling rate (0.01 °C min )1 ) was used for a small ice crystal. Figure 4A is a plot of TH activity measured for 0.1 mm nfeAFP8 after the annealing of an ice bipyra- mid for 0–3 h. The annealing was performed at a T m of )0.05 °Cor)0.25 °C. Each experiment used the same cooling rate of 0.20 °CÆmin )1 . The TH value Photomicroscope CCD Camera Object glas s Computer Temp. controlle r Display A B Crystal & Temp. monitor) LN 2 dewa r Stage Capillary holder Viewing hole Capillary (sample) Capillary Sample solution Oil Air Capillary holde r Freezing plate Fig. 1. Experimental set-up (photosystem) for viewing the growth and melting of ice crystal to determine thermal hysteresis value of an AFP solution. (A) The photosys- tem composed of a photomicroscope (Leica DMLB100), a temperature controller (Lin- kam THMS 600), a Colorvideo 3CCD camera (Sony), and an image-processing computer. (B) The capillary cell containing a 0.75 lL sample solution, and setting of the sample into the holder. 4.0 3.5 A B 0.5 1.0 1.5 2.0 2.5 3.0 0 0 0 .25 1.000.75 0.50 NaCl Glucose photo-system & osmometer ΔT f ( o C) Concentration ( M ) 20 μ m h g f e d c b a Fig. 2. (A) Concentration dependence of the equilibrium freezing point of NaCl and glucose solutions. Open symbols represent the data determined using the photosystem, and closed symbols are those obtained using a commercial osmometer (Vogel, model OM802). The error bars represent standard deviations of the data obtained from at least three experiments. (B) Snapshots showing the change in an ice bipyramid created in a 0.1 m M solution of type III AFP from Notched-fin eelpout (denoted nfeAFP8, cooling rate 0.01 °CÆmin )1 ). (a) T ¼ –0.46 °C; (b) T ¼ –0.61 °C; (c)–(h) T ¼ –0.81 °C. The nonequilibrium freezing point of )0.81 °C was evaluated from this experiment. M. Takamichi et al. Time-dependent enhancement of thermal hysteresis FEBS Journal 274 (2007) 6469–6476 ª 2007 The Authors Journal compilation ª 2007 FEBS 6471 tended to increase in proportion to the crystal anneal- ing time. When the ice bipyramid was annealed for 3 h at a T m of )0.05 °C, TH activity increased 1.4-fold compared with time zero. This TH increase reached 2.5-fold when the 3 h annealing was performed at a T m of )0.25 °C. Dependence of TH on the size of the seed ice crystal was also examined for 0.1 mm nfeAFP8 at a cooling rate of 0.20 °CÆmin )1 . The closed circles in Fig. 4B rep- resent TH values obtained after 2 h annealing of the ice bipyramid at a T m of )0.25 °C, and the open cir- cles, which are reproduced from Fig. 3C, depict com- parable data obtained without ice crystal annealing. TH decreased exponentially with increasing crystal size only after 2 h annealing at a T m of )0.25 °C, and no TH dependence on crystal size was evident when TH was measured soon after creation of the ice bipyramid. Discussion In the present study, we first examined the TH depen- dence of nfeAFP8 on the rate of cooling (Fig. 3), uti- lizing our in-house-built photomicroscope system. Data consistency between the system and a freezing- point osmometer (Fig. 2) validated the performance of the system for evaluating TH value. Using this system, we demonstrated that a slower rate of cooling ampli- fies TH, while a faster rate of cooling progressively diminishes TH until it reaches a plateau. A similar, but not identical, observation has been made for a small species of AFGP using a freezing-point osmome- ter; the AFGP exhibited significant antifreeze activity upon slow cooling, but lacked such activity if cooled quickly [18]. In addition, a time-dependent change in T f of a type I AFP solution has been demonstrated 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 0.1 0.2 0.3 0.4 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration (mM) 0.01 o C/min 0.20 o C/min 0 0.10 0.200.05 0.15 Coolin g rate ( o C/min) Thermal hysteresis ( o C) Thermal hysteresis ( o C) (slow) (fast) slow fast 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.01 o C/min 0.20 o C/min Ice crystal size ( μ m) 03515 252010 30 Thermal hysteresis ( o C) (slow) (fast) C A B Fig. 3. (A) Concentration dependence of TH for a 20 lm long ice crystal measured using 0.01 °CÆmin )1 (closed circles) and 0.20 °CÆmin )1 (open circles) rates of cooling. (B) Dependence of TH on the rate of cooling (°CÆmin )1 ). (C) Dependence of TH on the size of the ice crystal. Each point represents the mean of three experiments and the error bar represents the standard deviation. All experiments were performed immediately after preparation of an ice crystal in a 0.1 m M solution of nfeAFP8. Annealing time (h) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 1.0 2.0 3.0 Annealing Temp. = T m - 0.25 °C 1.4 2.5 1.0 Ice crystal size ( μ m) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 15 252010 30 35 Annealing Time = 2 h Annealing Time = 0 h Thermal hysteresis (°C) Thermal hysteresis (°C) A B Annealing Temp. = T m - 0.05 °C Fig. 4. Influence of annealing time and ice crystal size on TH. (A) Thermal hysteresis of nfeAFP8 measured after annealing of an ice bipyramid for a certain period of time (0–3 h). The measurement was repeated for three times at the annealing temperatures of T m )0.25 °C (closed circles) and T m )0.05 °C (open circles) using a cooling rate of 0.20 °CÆmin )1 . (B) Dependence of thermal hysteresis of nfeAFP8 on the size of the seed ice crystal. Closed circles are the data obtained after 2 h of annealing, and open circles are those without annealing. All other experimental parameters are the same as Fig. 3. Time-dependent enhancement of thermal hysteresis M. Takamichi et al. 6472 FEBS Journal 274 (2007) 6469–6476 ª 2007 The Authors Journal compilation ª 2007 FEBS using temperature-gradient thermometry [19]. No such time-dependent change in TH was reported for insect AFPs, while a linear and negative correlation was detected between TH and the logarithm of the mass fraction of ice crystal versus the total water mass for AFP from cerambycid beetle R. inquisitor [9,10]. We examined the dependence of TH on the crystal size under fast (0.20 °CÆmin )1 ) and slow (0.01 °CÆmin )1 ) cooling rates to clarify the correlation between the cooling rate and seed ice crystal size. These results show that TH decreases with increasing crystal size at the slow cooling rate, and does not depend on the crystal size at the fast cooling rate. These results imply that the cooling rate dominates the TH dependence on the seed crystal size. As actual tem- perature control of the freezing plate in our system is stepwise, a different cooling rate will produce a differ- ent time lapse of cooling on a sample. In other words, there is a longer annealing period before starting the TH measurement at a slower cooling rate. We there- fore examined whether an annealing period of 2–3 h influenced the TH value of nfeAFP8. An annealing experiment was performed within the hysteresis gap (0.25 and 0.05 °C below T m ), and TH was measured at a fixed cooling rate (0.20 °CÆmin )1 ). These parameters revealed that the annealing time amplifies TH, the amplification level is enhanced by lowering of the annealing temperature, and TH shows dependence on crystal size only after 2–3 h of annealing. As the time- dependent amplification of TH explains the detection of a higher TH value at the slower cooling rate, it may be concluded that the time of annealing is an essential influence on the TH value of nfeAFP8. For type III AFP, the 3D structure and the ice-bind- ing mechanism have been extensively examined. High- resolution X-ray and NMR structures have revealed that a remarkably flat and amphipathic surface is constructed on type III AFP, enabling complementary binding to the flat ice prism plane [14,21]. When the type III AFP solution contains a seed ice crystal, the AFP molecules come closer to the ice through the dif- fusion process, for which an association constant of 10 7 )10 8 s )1 Æm )1 can be assumed from the Smulochow- ski model [23]. The AFP molecules then undergo prompt binding onto the flat ice plane, which has been thought to progress irreversibly according to the adsorption–inhibition mechanism at the ice–water interface [3,4]. Subsequently, convex ice fronts are formed between the ice-bound AFPs on the flat plane, as depicted in Fig. 5A. At this stage, the ice crystal adopts a bipyramidal shape. During subsequent TH measurements, the convex ice front overgrows (Fig. 5B, dashed line), allowing uncontrolled growth at the nonequilibrium T f . This leads to the detection of a nonzero TH value at time zero (0 h of annealing). The height and curvature of the convex ice front are defined by spacing between adsorbed AFP molecules (i.e. the Kelvin effect [24]), such that the TH value reflects the fraction of ice-bound AFPs on the flat ice plane. Indeed, TH increases in proportion with the total concentration of nfeAFP8, as can be seen in Fig. 3A. The fraction of AFPs on the flat ice plane is not, however, changed during the present time-depen- dent experiment on TH (Fig. 4) as we fixed the con- centration of AFP at 0.1 mm. A plausible explanation for the time-dependence of TH is that nfeAFP8 under- goes ‘secondary’ binding onto the convex ice front over a very slow time scale (2–3 h; Fig. 5C). Indeed, a recent study on ice etching revealed that type III AFP can bind to several ice planes in addition to the (10– 10) prism plane [14]. TH is the level of supercooling required to nucleate ice growth from the ‘weak’ convex ice fronts, the growth of which is not strongly inhib- ited by AFP. If AFP undergoes secondary binding C AFP low TH TH measurement A annealing time (2–3 h) TH measurement B D solution ice ice ice high TH ice Fig. 5. Schematic drawing of ice growth inhibition of type III AFP based on the ‘adsorption–inhibition’ model [3,4]. (A) A convex ice front is created after primary binding of type III AFP onto the flat ice plane. (B) Overgrowth of the convex ice front, giving a low TH value. (C) Possible secondary ice binding of type III AFP onto the convex ice front during the annealing period (2–3 h). (D) AFPs bound onto the convex ice front narrow the growth area (dashed line), leading to an enhancement of TH activity. M. Takamichi et al. Time-dependent enhancement of thermal hysteresis FEBS Journal 274 (2007) 6469–6476 ª 2007 The Authors Journal compilation ª 2007 FEBS 6473 over a very slow time scale after the primary ice bind- ing, it stabilizes the convex ice front (Fig. 5D), leading to the time-dependent change in TH activity. This con- cept further explains the increased level of time-depen- dent amplification of TH at the lower annealing temperature (Fig. 4A). The convex ice front will become more accentuated by lowering the annealing temperature, which will raise the opportunity for bind- ing of AFPs to the growing convex ice front, thereby enhancing TH activity. We recently monitored the flu- orescence of seed ice crystals at 30 min intervals in a solution of a fusion protein comprising nfeAFP8 and green fluorescence protein (nfeAFP8–GFP) (see supple- mentary Fig. S1). We observed a slight increase of fluorescence intensity in a region proximal to the seed ice surface, accompanying a slight growth of ice crystals in proportion with the annealing time (0–3 h). It is noteworthy that such an intensity change was observed only at the lower annealing temperature (T m )0.25 °C). These preliminary data are also consis- tent with the idea of progressive binding of AFP over a time scale of 2–3 h. A larger number of convex ice fronts are presumably located on a larger-sized ice crystal. If secondary binding of AFP has the ability to stabilize convex ice surfaces, such stabilization becomes imperfect with an increasing number of weak points on the ice crystal. This supposition is consistent with the data obtained after 2 h of annealing (Fig. 4B, closed circles), for which a reduction in TH was apparent with increasing size of the ice crystal. The lack of significant size dependence of TH activity (Fig. 4B, open circles) might be ascribed to nonstabilization of the weak point of any size of the crystals, because of the lack of secondary binding of AFP at time zero (0 h of anneal- ing). It should be noted that no apparent time-depen- dent TH increase was detected for different types of AFP (type I AFP from great sculpin and type II AFP from Japanese smelt; data not shown). Therefore, it is not clear whether such a slow accumulation process is a general mechanism for any type of AFP. Several past studies have shown that the bipyramidal ice crystal can be maintained for a long time [25], but no TH data have been reported for such an ice bipyramid. We believe that a simple TH measurement before and after 2 h will enable us to learn more about the secondary binding of AFP over a very slow time scale, which will be detected as enhancement of the observed TH value. To summarize, we have successfully prepared a recombinant type III AFP isoform and carefully mea- sured its TH activity using an in-house-built photomi- croscope system. Measurement of TH dependence on various experimental parameters revealed, for the first time, that annealing time significantly causes an enhancement of TH activity of fish type III AFP. The adsorption–inhibition model may explain this time- dependent change in TH if type III AFP undergoes sec- ondary binding to the convex ice front following bind- ing to the primary ice plane. Experimental procedures Sample preparation of AFPIII A sample of the type III AFP isoform nfeAFP8 was pre- pared for TH measurements as described previously [20] with the following modifications. A soluble fraction con- taining a recombinant nfeAFP8 after sonication of Escheri- chia coli BL21 (DE3) was dialyzed against 50 mm citric acid buffer (pH 2.9). After dialysis, cation-exchange chro- matography was performed using an Econo-Pac High S cartridge (Bio-Rad, Hercules, CA, USA) with a linear NaCl gradient (0–0.5 m)in50mm sodium citrate buffer (pH 2.9). The purified nfeAFP8 was concentrated (approximately 7mgÆmL )1 ), and then diluted prior to TH determination. For all the TH experiments, the sample was dissolved in 0.1 m ammonium bicarbonate (pH 7.9). TH measurement system The experimental set-up using a commercial photomicro- scope system (denoted as the ‘photosystem’) is illustrated in Fig. 1. The two main instruments in this system were a Leica DMLB100 photomicroscope (Leica Microsystems, Wetzlar, Germany) and a Linkam THMS 600 temperature controller (Linkam Scientific Instruments Ltd, Tadworth, Surrey, UK) equipped with a liquid nitrogen Dewar flask. The latter con- trolled the temperature of the freezing plate with an accuracy of 0.01 °C by combining the use of an electric heater and liquid nitrogen. The left portion of Fig. 1(B) illustrates the position of the sample solution in a quarter of a Hirschmann capillary tube (Hirschmann Labogera ¨ te, Heilbronn, Ger- many) (length 30 mm, diameter 0.92 mm); each end of the tube was sealed using mineral oil (approximately 1 lL each) to prevent vaporization of the sample solution (approxi- mately 0.75 lL). The sample-containing capillary was then loaded into a custom-made copper capillary holder (diameter 17 mm, thickness 2.5 mm) (Fig. 1B, right), which was placed into the freezing plate on the cooling stage (Fig. 1A). After positioning of the capillary tube, 15 lL of ethylene glycol was poured through the viewing hole (Fig. 1B, right) to achieve thermal conductivity between the holder and capil- lary tube. The ice crystal image was captured using a Color- video 3CCD camera (Sony, Tokyo, Japan) (Fig. 1A), and the temperature status was simultaneously viewed on a dis- play and saved as a video file on a personal computer (Fig. 1A). We determined T f and T m by monitoring a slower Time-dependent enhancement of thermal hysteresis M. Takamichi et al. 6474 FEBS Journal 274 (2007) 6469–6476 ª 2007 The Authors Journal compilation ª 2007 FEBS playback of the video. The size and length of the ice bipyra- mid was evaluated from the crystal image captured by the video. For accurate determination of the crystal length, we initially captured the image of a 30 lm fiber inserted into the sample solution, and found that the captured length needed to be multiplied by 1.2 to determine the actual length of the crystal. Measurement of the T f and T m values was repeated at least three times, and the values were averaged. More details of the TH measurement procedure are described in Results. A model OM802 commercial freezing-point osmometer (Vogel, Giessen, Germany) was used to check the perfor- mance of the photosystem. We initially placed a 50 lL sam- ple into the cooling bath ()7 °C) of the osmometer. When the sample temperature reached )7 °C, the instrument automatically inserted a frosty probe into the sample solu- tion to initiate the growth of ice crystals. Accordingly, latent heat was emitted owing to the phase transition from liquid to solid state, which raised the temperature of the sample. After completion of the rise in temperature, an ice– water equilibrium state was achieved at a certain negative temperature, which was the T f value in this instrument. Before performing measurements, the osmometer was nor- malized with a standard fluid of 300 mOsmol. Acknowledgements We are grateful to Brian Sykes at the University of Alberta (Edmonton, Canada) for fruitful discussions on the ice-binding mechanism of nfeAFP8. References 1 Jia Z & Davies PL (2002) Antifreeze proteins: an unusual receptor–ligand interaction. Trends Biochem Sci 27, 101–106. 2 Davies PL & Hew CL (1990) Biochemistry of fish antifreeze proteins. 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Cryo-Lett 14, 31–36. 25 Haymet ADJ, Ward LG, Harding MM & Knight CA (1998) Valine substituted winter flounder ‘antifreeze’: preservation of ice growth hysteresis. FEBS Lett 430, 301–306. Supplementary material The following supplementary material is available online: Fig. S1. Time-dependent changes in photomicroscope image of an ice bipyramid in the solution of nfeAFP– GFP. This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article. Time-dependent enhancement of thermal hysteresis M. Takamichi et al. 6476 FEBS Journal 274 (2007) 6469–6476 ª 2007 The Authors Journal compilation ª 2007 FEBS . Effect of annealing time of an ice crystal on the activity of type III antifreeze protein Manabu Takamichi 1,2 , Yoshiyuki Nishimiya 1 , Ai Miura 1 and. results in the TH dependence on the annealing time. Abbreviations AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP8, an isoform of type III AFP

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