In Situ Observation of Antifreeze Glycoprotein Kinetics at the Ice

Sep 12, 2008 - ABSTRACT: Antifreeze glycoproteins (AFGPs) are a necessary tool for ... Here, we study the adsorption kinetics of AFGPs during solution...
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In Situ Observation of Antifreeze Glycoprotein Kinetics at the Ice Interface Reveals a Two-Step Reversible Adsorption Mechanism Salvador Zepeda,† Etsuro Yokoyama,‡ Yukihiro Uda,† Chihiro Katagiri,† and Yoshinori Furukawa*,†

CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 10 3666–3672

Institute of Low Temperature Science, Hokkaido UniVersity, Kita-19, Nishi-8, Sapporo, Japan 060-0819, and Computer Center, Gakushuin UniVersity, 1-5-1 Mejiro, Toshima-ku, Tokyo, Japan 171-8588 ReceiVed March 14, 2008; ReVised Manuscript ReceiVed June 12, 2008

ABSTRACT: Antifreeze glycoproteins (AFGPs) are a necessary tool for the survival of fish that live in subfreezing environments [Yeh, Y.; Feeney, R. E. Antifreeze proteinssStructures and mechanisms of function. Chem. ReV. 1996, 96 (2), 601-617]. Although scientists agree that these proteins arrest ice crystal growth by a surface adsorption mechanism, the exact nature of the interaction remains an open question. Here, we study the adsorption kinetics of AFGPs during solution ice crystal growth using confocal fluorescence microscopy within and just below the freezing-melting temperature hysteresis region. The AFGP kinetics at the ice surface reveal a two-step inhibition process: (i) incomplete adsorption or a weak interaction that modifies the surface for (ii) a stronger interaction to achieve the complete adsorption necessary to halt growth. The growth is modified from a rough interface to a faceted one, and growth is halted at supercoolings less than 0.05 °C. However, growth resumes, and the proteins desorb, return to the solution phase, and are not incorporated into the ice crystal as previously proposed. Our findings are contrary to an AFGP mechanism described by the Gibbs-Thomson model. We argue that an alternative explanation must include a solvated protein interacting with a solvated ice surface. While thermodynamics considerably alter the interfacial region, antifreeze action is a purely kinetic phenomenon. Introduction Since the discovery of the “antifreeze effect”, which allows fish to survive in supercooled waters, by Scholander et al. over 50 years ago,1 many antifreeze proteins (AFPs) and glycoproteins (AFGPs) have been discovered in fish,2–4 insects,5,6 plants, and bacteria7,8 and have been explored as cryosurgery adjuvents9 and human organ preservatives10 and are even used as frozen foods additives.11 There exist at least five distinct classes ranging in structure from a short R-helical rod to extended helices and even larger globular forms with distinct compositions; yet, they all accomplish the same basic functions: inhibit ice crystal growth and ice recrystallization and radically modify the growth forms. The amount of AFGPs required for the freezing inhibition is up to 500 times less than that of the colligative effect12 and even much less for some insect proteins.5,6 This has led authors to propose pinning models13 based on hydrogen bonding between the protein surface and the crystal facet or steps poisoning that location from further growth, the GibbsThomson model.14-16 The new freezing point is determined by changes in the surface free energy due to local curvature changes from overgrowth between surface-bound proteins. Recently, the focus has turned to hydrophobic interactions rather than the hydrogen bonding itself. One of the main drawbacks of these proposed models, where the protein snuggly fits on the surface, is that the ice surface is characterized as an abrupt termination of the bulk crystal structure with an ice/vacuum interface. That is, no surface reconstruction is considered, nor is the surface melting or quasiliquid layer (QLL), which is known to be most extensive near the melting point. In fact, the surface liquid largely contributes to such interactions and can play a dominant role, as seen in * To whom correspondence should be addressed. Tel/fax: +81-11-706-5467. E-mail: [email protected]. † Hokkaido University. ‡ Gakushuin University.

other model systems.17 Additionally, in recent computer simulations for the type I AFP at the ice/water interface, the interaction was seen to take place with the hydrophobic side of the protein facing the ice, but the protein was found well within the QLL.18 The extensive QLL near the melting point can be thought of as a diffuse “surface reconstruction”, leaving no well-defined surface for the protein to bind to but rather a rough interface at the molecular level. However, it is well-known that ice growth in the presence of all types of antifreeze promotes highly faceted gross morphologies. Furthermore, the similar previously proposed direct hydrogen-bonding mechanisms for the type I AFPs, a nonglycosylated AFP with a well-defined R-helical structure, and regularly spaced threonine residues, have been disproved by several authors using mutagenesis techniques.19-21 The residues that were thought to be involved with the hydrogen bonding were replaced with hydrophobic residues, and antifreeze action similar to the native protein was still observed in the mutants. Again, this suggests that the hydrophobic residues play a much larger role than previously thought, although other residues are still available for hydrogen bonding via other motifs.22 Unlike the nonglycosolated AFPs, the AFGPs are known to be quite flexible,23,24 leaving no well-defined surface on the protein. While this may provide an energetic advantage, there remains the question of the highly fluidic nature of the ice surface for a tight binding mechanism as previously envisioned.25 Nevertheless, it has been argued that the protein binding is permanent and that the protein’s strong affinity for the ice surface would result in the proteins incorporating into the ice crystal matrix upon further growth. Knight et al. have measured the amount of AFGP7,8 that incorporates into crystalline ice hemispheres grown into AFGP solution from a coldfinger to be about 10-15%.25 In similar experiments, Marshall et al. measured a partitioning coefficient close to unity for type II and type III fish AFPs as well as two insect AFPs.26 However, in the latter study, the ice samples were polycrystal-

10.1021/cg800269w CCC: $40.75  2008 American Chemical Society Published on Web 09/12/2008

AFGP Kinetics at the Ice Interface

line, and the partitioning should include proteins that are trapped between grain boundaries and any inside the crystal, as suggested by Knight et al.25 Such grain boundary inclusions were clearly seen by one-directional ice crystal growth in the AFGP-8 solution by Zepeda et al.,27 while no detectable amounts of protein were found inside the crystal. However, all of these experiments measure phenomenon during growth under large temperature gradients, in some cases even under kinetic roughened growth conditions, whereas the primary function of many of these proteins is to inhibit growth to allow survival of the species at very moderate supercoolings, less than 1 K in the case of most fish, where the growth and final morphology during inhibition is known to be smooth. Additionally, the ice surface characteristics can vary quite dramatically, especially near the melting point where these proteins function. Although the overall consensus is that the antifreeze effect is a surface phenomenon, a clear description of the protein kinetics for the antifreeze at the ice surface is lacking and is essential to understand the mechanism. In this work, we directly show that for an active sample of the AFGP, binding is reversible. Here, we directly observe the protein kinetics at the ice surface by following the proteins during growth and during inhibition. To achieve this, we labeled a mixed sample of AFGP 4-6 (AFGP1-8 varying in size from 2.6 to 33 kDa) with fluorescein isothiocyanate (FITC),27 grew single ice crystals from a capillary into a 5 µg/mL AFGP solution, and used laser scanning confocal microscopy for imaging. Materials and Methods Antifreeze Glycoproteins. Each AFGP consisted of a varying number of repeating units of alanine-alanine-threonine, with minor sequence variations and the disaccharide β-D-galactosyl-(1,3)-R-Nacetyl-D-galactosamine joined as a glycoside to the hydroxyl oxygen of the threonine residue, varying in molecular mass from 2.6 to 33 kDa, AFGP1-8. A mixture of the AFGP4-6 used in this study, a gift from Professor Yin Yeh (University of California, Davis), was isolated from the blood sera of trematomas borchgrevinki. Roughly 70% was fraction 6, 20% was fraction 5, and 10% was fraction 4, as determined by measuring the absorbance at 280 nm after separation by highperformance liquid chromatography (HPLC). The proteins were labeled at the N terminus with FITC (Pierce Biotechnology, Rockford, IL) with a 24-fold excess molar ratio. The labeling reaction was carried out at pH 8.5. Excess FITC was removed by using a dextran desalting column (D-Salt Dextran Desalting Columns, Pierce Biotechnology). The labeled protein was then dialyzed (Slide-A-Lyzer 2K MWCO, Pierce Biotechnology) for at least one day against filtered and deionized water to remove buffer salts. Although the dialysis membranes were sufficient to remove the excess FITC and buffer salts, the dimethylformamide used to reconstitute the FITC powder was incompatible with the membranes. Ice Crystal Growth and Imaging. Single ice crystals were grown in solution from a glass capillary. Capillary growth ensured that we obtained a single ice crystal oriented with the c-axis normal to the capillary. The growth cell was similar in concept to that described elsewhere28 and references with modifications that allowed for imaging with an inverted microscope (TE 2000-E, Nikon Tokyo, Japan) fit with a spectral imaging confocal laser scanning system (C1si, Nikon Tokyo, Japan). Briefly, the growth cell was designed to nearly eliminate temperature gradients and allow for manipulation of the single crystal during growth. The central piece was machined from a 40 mm cube of oxygen-free copper. The growth chamber (Figure 1) was a cylinder of 10 mm diameter and 10 mm in height strategically placed so that the crystal could be imaged within the working distance of a 10× objective (Nikon, PH1 DL). A triple window system with O-ring spacers was used to minimize temperature gradients. The temperature was controlled with thermoelectric elements controlled by a PID system (MTCA15100, Melcor Corp., Trenton, NJ) with copper heat sinks connected to a circulating bath held at 10 °C. The thermoelectric elements were placed on two opposite sides of the growth cell. The temperature was controlled to within 2 mK inside the copper mass, while the best

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Figure 1. Growth cell. The temperature was controlled with a thermistor within the main body of the copper growth cell (a) and measured inside the solution (b). Ice was nucleated at the capillary (c), and a single ice crystal typically with the c-axis perpendicular to the capillary reached the solution. conditions yielded less than 5 mK radial variations across the solution with the warmest spot at the center. Ice was nucleated by cooling the capillary with cold liquid ejected from a commercially bottled air duster by spraying with the bottle inverted. To prevent ice from nucleating on the sidewalls of the growth chamber, the capillary was insulated with a Teflon capillary holder. In general, only one ice crystal oriented with the a-axis parallel to the capillary reached the solution. The capillary holder was then rotated to adjust the c-axis orientation. Protein Distribution Calculations. Figure 2a shows a typical image of an ice crystal growing into AFGP solution. The intensity as seen in the image is a direct measure of the number of fluorescent molecules within the field of view, since the intensity is directly proportional to the number of molecules. Figure 2b shows the calculated average intensity along the length of the blue rectangle. Icap is equivalent to the background intensity, and Isoln determines the proportionality between the intensity and the concentration allowing the conversion from intensity to concentration as shown in Figure 2c. However, this represents the signal contribution throughout the entire z-field of view, and we are interested in measuring the protein adsorbed on the surface and included inside the crystal. Because we are using a long working distance objective, it is not possible to measure z-sections of the thin ice crystal as oriented in Figures 2 and 3. However, z-sections are possible by rotating the crystal 90° as shown in Figure 4, allowing us to measure the concentration inside the crystal. Here, we see that the intensity inside the crystal to within error is nearly the same as inside the capillary. We will discuss the trace amounts later. To calculate the amount of adsorbed protein, we must consider the contributions to the signal from the solution molecules as well as the protein included inside the crystal to determine the proportionality between I and C per molecule. The intensity due to the volume of solution displaced by the thickness of the crystal is ∆I ) Isoln - Iice - Iinice, where Iinice is the contribution from protein inside the crystal and can be estimated from Figure 4, although here this can be ignored. The average intensity per molecule is given by ∆I divided by the number of molecules in the volume displaced by the crystal, calculated from the known concentration. Finally, we can convert the intensity from Figure 2b to molecular density (mol/area) and subtract the solution contribution, taking into account the thickness of the ice crystal and the pyramidal orientation at the interface. The pyramidal plane angle is roughly 75°, and the plotted intensity is the average intensity/pixel (1 pixel ) 1.06 µm). Figure 5 shows the final result, that is, only the surface-adsorbed species across the interface. The four central points were used to calculate the molecular spacing.

Results and Discussion Figure 2 shows an ice crystal growing in AFGP4-6 solution of concentration c ) 5 µg/mL at a supercooling of less than ∆T ) 0.05 °C (Supporting Information). The interface is modified to a high indexed pyramidal plane as seen with the AFGPs as well as with other AFPs. However, the concentrations

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Figure 2. Protein concentration at the ice surface. (a) Confocal image of an ice crystal with protein adsorbed on the stopped interfaces and no adsorption at the single growing interface. Graph b shows the average intensity along the length of the blue rectangle in panel a. Icap, Iice, and Isoln are the intensities of the capillary, ice region, and solution, respectively. Icap is equivalent to the background intensity, and Isoln determines the proportionality between intensity and concentration, allowing the average concentration calculation in graph c. The halted faces show a large quantity of adsorbed protein seen as the large peak in graph b. In contrast, the growing interface in graph c shows no protein in addition to the solution at the interface. The inset in panel a shows a phase contrast image of an ice crystal grown in pure water.

Figure 3. Adsorption and desorption during inhibition and growth. Images were taken 9, 13, and 18.6 s after Figure 2a. The concentration along the interface, the blue dashed line in panel a, is shown graphically as the solid blue line in graph d. The black line is the fit to the data as a guide. At the arrows in panel a, a new surface along the halted interface is formed. Growth continues in the [011j0] direction, and when the interface reaches the apex, growth is halted and proceeds in the [11j00] direction, as in panel b. Protein adsorbs on the (101j0) face, while the protein on the (11j00) face desorbs during growth. (e) Average intensities along the length of the rectangles in panels a-c, color-coded accordingly. The plots show adsorption at the interface (black), desorption and protein diffusing into the solution (gold), and protein distribution during growth (red) as in Figure 1c with the interface concentration at solution level. Additionally, in panel c, where the peak was in panel a, the concentration drops to levels as the rest of the solid showing removal of the adsorbed protein.

AFGP Kinetics at the Ice Interface

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Figure 5. Adsorbed AFGP. The average protein density across the interface is calculated from Figure 1b. (a) Plot showing the contribution to the signal from the adsorbed protein. (b) Plot before removal of the solution contribution to the signal and before correcting for the width and tilt of the pyramidal plane. The ice is to the left of the peak and the solution is to the right.

Figure 4. Amount of protein inside the crystal. The crystal was rotated 90° with respect to the images in Figures 1 and 2 to obtain a clear section of the solid. The graphs in b and c represent the average concentrations along the red and blue rectangles without the background subtraction. Some edge effects are seen in graph b, but in the center, the intensity drops to the background levels, 0.25 ( 0.08 µg/ml, as measured in graph c along the glass capillary, 0.16 ( 0.07 µg/ml.

used here are 3 orders of magnitude smaller than studies where the ice crystal takes on a complete bypyramidal shape. Here, the growth morphology tends to more closely resemble the pure water growth forms (inset of Figure 2a) even at the higher supercoolings, but the rough interface becomes smooth. That is the normal growth of the ice crystal in pure water switches to a layer-by-layer growth mode in the AFGP solution. When the crystal exits the capillary, all sides are growing, but eventually, only one side continues to grow into the supercooled solution. Here, we are very close to the freezing temperature, and only a small variation in temperature can move the conditions to within the hysteresis gap where the growth is inhibited. This is possible since heat released during growth can elevate the temperature and halt the growth, while only the

facet that is far enough into the supercooled region continues to grow. Eventually, the halted interfaces reach the bulk supercooled solution temperature, but these do not continue to grow since protein adsorbs on the surface and lowers the local freezing temperature below the solution temperature. Growth Modification. Figure 2b,c shows the protein distribution across the halted and the growing interfaces. The growing interface does not show a significant amount of adsorbed protein (Figure 2a,c); nevertheless, there is a strong enough interaction to radically modify the growth form as compared to the pure water crystal (inset of Figure 2a), even at these relatively small concentrations. At this interface, the temperature is higher than at the halted interfaces, since latent heat that is released during growth slightly increases the temperature and a higher supercooling due to local temperature variations cannot explain the continued growth. Instead, it is more likely that complete adsorption does not take place due to the protein dynamics that can be slower than the growth dynamics at this interface. Using NMR and FTIR techniques, it was determined that the proteins are quite flexible in solution29 and remain so even at temperatures as low as -80 °C, but upon adsorption, they take on a slightly more ordered structure,23,30 and it is this conformational change that can slow down the adsorption process and, hence, the growth inhibition process altogether. The surface modification and protein conformation changes are the first step toward the final adsorption step that completely halts the growth, indicating a multiple step mechanism. Surface Coverage. The stopped interfaces show several features that are distinct from the growing interface. A significant amount of protein adsorbs at these interfaces relative to that in solution. Figure 2b shows a large peak in intensity at the interface that corresponds exactly to the adsorbed quantity. The spacing between molecular centers is d ) 21 ( 4 nm, nearly identical to previous measurements for type III31 (d ) 20 ( 5 nm, c ) 0.3-3 mg/mL) and type I AFPs32 (d ) 4-20 nm, c ) 2 mg/mL), despite very different solution concentrations that lead to very different freezing temperature suppression, ∆T. If we consider the size of the AFGPs using an estimate based on length and number of the tripeptide units33 and assume that the proteins adsorb to the ice interface in equal proportions to that in solution, the surface coverage is approximately θ ) 0.04.

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Additionally, if we consider that only AFGP4 adsorbs, the most active band, the coverage is approximately θ ) 0.06. This reduces the protein spacing to 17 and 16 nm for the former and latter, respectively. The Gibbs-Thomson model predicts a freezing temperature lowering due to surface free energy changes determined by the overgrowth between adsorbed species given by

∆T )

2ΩγTO F∆HO

(1)

where Ω is the molar volume of ice, γ is the surface free energy, ∆HO is the latent heat of fusion, and 2F ) d is the spacing between the adsorbed molecules. For a spacing of d ≈ 17 nm, the Gibbs-Thomson model predicts a freezing temperature lowering of 6 °C. This value is several orders of magnitude lower than the experimental solution temperature. However, the AFGP sample used contains both active and inactive species. The larger AFGP1-5 are known to suppress the freezing temperature, while AFGP6-8 show no appreciable activity.34 If we assume that all of the AFGPs adsorb in equal proportions and consider only the most active of the adsorbed species in calculating the freezing temperature lowering, that is, only consider the spacing between adsorbed AFGP4 molecules, d ≈ 170 nm, the G-T model would predict a freezing temperature of 0.6 °C, which is still 1 order of magnitude larger than the experimental temperature. Grandum et al.32 calculated a freezing temperature lowering of 0.2 °C for a spacing of 10 nm using this model. The large discrepancy with their value is due to the difference in value for the surface free energy used. Here, we used the value of 30 erg/cm2, whereas in their calculations, a value of 2 erg/cm2 was used. An estimate for the surface free energy was obtained by counting the broken bonds and gave 14 and 15 erg/cm2 for the basal and prismatic faces35 and experimental values range between 29 and 44 erg/cm2 for the ice/water interface.36-39 AFGP Adsorption. As the ice front advances in the growth direction, the neighboring halted planes expand in surface area. Looking carefully at this new area on the halted interfaces (arrows in Figure 3a) reveals that the adsorption process does not occur instantly. In fact, it can take several seconds to reach the maximum surface coverage seen here (Figure 3d). However, during the adsorption process, the interface does not propagate, indicating that the maximum experimentally observed coverage is not necessary to halt the growth or that the slightly increased interface temperature due to the crystal growth will significantly decrease the amount of protein required to keep the expanding interface from propagating. The latter does not explain the subsequent growth despite the overadsorption of AFGP that is sufficient to halt the growth according to the G-T model. Thus, we consider that only the first molecules that are adsorbed are necessary to halt the growth and refer to these as strongly adsorbed molecules, while weakly adsorbed molecules subsequently fill in the remaining surface (Figure 6a). This is similar to a kinetic pinning model proposed by Sander and Tkachenko,40 except that they consider the overadsorbed proteins to bind irreversibly, whereas we observe reversibly bound proteins. However, we reemphasize that at the observed surface coverage the G-T model predicts that the interface should not resume growth as seen. Additionally, the Gibbs-Thomson model by nature will yield a rough interface; clearly, this is not the case with the AFGPs or other AFPs that are all known to promote faceted growth in the form of hexagonal bypyramids. AFGP Desorption. When growth resumes on the halted planes, the ice growth front releases the proteins from the

Figure 6. AFGP at the ice surface. The dark blue region represents the solid, while the light blue region represents the liquid. (Top) Red ovals represent the strongly bound AFGP necessary to halt the growth, while the gray ovals represent weakly bound AFGP that do not play a critical role in the initial freezing suppression. (Bottom) An alternative view that depicts AFGP adsorbed in a diffuse ice/water interface. In the latter, the protein is seen disrupting the transient region at the ice interface, effectively disrupting the water molecule’s path from the liquid phase to the solid phase.

surface, indicating that the adsorption is weak enough that the addition of water molecules can eject the proteins from the surface (Figure 3a-c,e). This continued growth is in contradiction with the Gibbs-Thomson model since the number of adsorbed molecules should be more than sufficient to keep the interface from growing. Additionally, no detectable amounts of the protein are seen incorporated into the crystal itself as would be expected if the proteins were irreversibly bound (Figure 4). Recently, Zepeda et al. showed that AFGP8, the shortest band of the AFGPs, exhibits similar rejection during growth.27 This relaxes the irreversible binding requirement that was once thought necessary for the AFGPs. In contrast with similar confocal fluorescent experiments, Pertaya et al. observed no mobility of type III AFPs,31 a nonglycosylated globular AFP, at similar protein spacings as seen here. Clearly, the reversible binding of the AFGPs indicates a much weaker interaction and a more dynamic process between the proteins and the ice interface than once thought. Surface Liquid Role. Alternatively, we can consider a thermodynamically driven interaction rather than a kinetic one. The AFGPs are highly flexible with no single prevailing conformation,29 and this makes them unique from the rest of the AFPs whose structures are more rigid. Similarly, the surface of ice is highly dynamic and anisotropic and in models is seen to have a transient region that extends up to nanometers between the solid and the liquid where the properties gradually change from one phase to the other.41,42 The proteins affinity is greatest for the prismatic plane43 whose transient region is largest and can provide an environment where the most favorable conformation can be achieved. All of the AFPs have a very high content of hydrophobic residues, and these do not necessarily get shielded from water in the folded state and are available to help stabilize or balance the protein near the surface in conjunction with the hydrogen bonding (Figure 6b). While the H-bonding between the protein surface and the rigid bulk ice should be a fundamental part of the interaction, the solvation

AFGP Kinetics at the Ice Interface

of both surfaces also plays an important role if not the dominant one. Recently, Noy et al. measured the interaction force between two model hydrogen-bonding surfaces of relevant dimensions in a polar solvent and showed exactly this, that the solvation entropy not only contributes but can indeed dominate the process.17 Thermodynamically, the change to a layer-by-layer growth mode indicates an increase in the surface free energy when compared to the pure water system. It is known that type III AFPs lower the surface free energy of water.44,45 Furthermore, it is argued that a similar change in the surface free energy by AFGPs results in a 3-fold increase in the thickness of the QLL at the ice/solution interface.30 However, with a lowering of the surface free energy, we would expect the opposite observation, that is, a smooth interface to become rough. However, in fact, the smooth ice interface created by any of the AFPs does not thermally roughen. The thermodynamic changes achieved by the AFGP while significant do not explain the morphological changes. Instead, the “antifreeze effect” must come from purely kinetic means, but rather than blocking surface sites directly on the crystal lattice, we can expect the proteins to tie up sites within or disrupt the quasi-ordered fluid. In previous pinning models, the protein solvation and interaction with the transient region water molecules are ignored altogether. Summary and Conclusions Several key observations lead to the conclusion that the Gibbs-Thomson model does not describe antifreeze action of the AFGPs. (i) At low concentrations of AFGPs, ice becomes faceted, indicating a change to layer-by-layer growth. The G-T model necessarily would produce a rough interface. (ii) After growth is stopped, protein adsorption continues. For the final coverage, the G-T model predicts a 6.8 °C freezing temperature lowering. Growth resumes despite the large amounts of adsorbed proteins. (iii) AFGPs do not incorporate into the ice crystal as would be expected; instead, they are released during regrowth. (iv) AFPGs are reversibly bound to ice. Taking into account both the flexible nature of the protein and the highly dynamic ice surface, rather than a rigid match between a surface determined by direct cleavage of the bulk structure, a more realistic interaction to consider is one where the AFGPs disrupt the quasi-ordering of the surface liquid at the interface by purely kinetic effects. Acknowledgment. We thank Y. Yeh (University of California, Davis) for providing the AFGP sample, Shuichiro Matsumoto (Hokkaido University) for assistance with the AFGP labeling, Shunichi Nakatsubo for assistance with the growth cell design, and the Nikon Imaging Center (NIC) at Hokkaido University for facilities use and confocal imaging assistance. This research was partially supported by the Ministry of Education, Science, Sports, and Culture, Grant-in-Aid for Scientific Research (Grant 18204036), and also by the Grant of the Ground Research for Space Utilization promoted by the Japan Space Forum (JSF). S.Z. thanks the Japanese Society for the Promotion of Science (JSPS) for their support. Supporting Information Available: Time-lapse video of ice crystal growth in 5 µg/mL AFGP4-5 solution at T < 0.05 °C. Video is 3× real time. This material is available free of charge via the Internet at http://pubs.acs.org.

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