Growth Habit Modification of Ice Crystals Using Antifreeze

DOI: 10.1021/cg1005083

Growth Habit Modification of Ice Crystals Using Antifreeze Glycoprotein (AFGP) Analogues

2010, Vol. 10 5066–5077

)

Raoul Peltier,†,§ Clive W. Evans,‡ Arthur L. DeVries,^ Margaret A. Brimble,†, Andrew J. Dingley,†,‡ and David E. Williams*,†,§

Department of Chemistry, ‡School of Biological Sciences, §MacDiarmid Institute for Advanced Materials and Nanotechnology, and, Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand, and ^Department of Animal Biology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin, Urbana, Illinois 61801, United States )

†

Received April 16, 2010; Revised Manuscript Received September 17, 2010

ABSTRACT: The research reported here considers the modification of ice crystal growth habit using analogues of antifreeze glycoproteins: compounds that control the growth of ice crystals in the blood of Antarctic fishes. A range of analogues of the smallest antifreeze glycoprotein (AFGP8: Ala-Ala-Thr-Ala-Ala-Thr-Pro-Ala-Thr-Ala-Ala-Thr-Pro-Ala) were synthesized, all of which have either N-acetyl-galactosamine or galactose moieties attached to their threonines instead of the native galactose-Nacetyl-galactosamine disaccharide. We also synthesized analogues in which the prolines were substituted with alanines in the protein sequence. The analogues were systematically studied for their effects on ice crystal shape and their effects when combined with a range of salts chosen across the Hofmeister series. A simple 1H NMR freezing experiment was used to detect adsorption onto ice and indicate differences in water proton hydrogen bonding. CD spectroscopy highlighted the role of the terminal galactose, the proline residues, and the N-acetylamine group in modifying the solution conformation. The results illustrate a delicate balance between the effects of hydrophobic and hydrophilic groups on the glycoprotein and the interactions between the glycoprotein, a developing ice crystal, and water. We demonstrate three ways of modulating the ice crystal shape: by changing the adsorbent concentration, by changing the adsorbent structure, or by altering the interaction of the glycoprotein with liquid water through the use of simple solution additives. We show a continuum of behavior between no shape modification and no thermal hysteresis to a strong shape modification and a significant thermal hysteresis, and we relate the results to kinetic models for antifreeze activity.

Introduction Ice exists in different crystalline and amorphous forms. However, hexagonal ice (ice Ih) is the most common crystal form, since it is the only one stable under normal pressure at 0 C.1 With the development of cryopreservation technologies2-4 and advances in cryosurgery,5 understanding ice crystal growth control has become increasingly important. A biomimetic approach to ice crystal growth modification is proposed here, based on the presence in Antarctic notothenioid fishes of antifreeze glycoproteins that modify and control the growth of ice crystals.6-9 Antifreeze glycoproteins (AFGPs) and/or antifreeze proteins (AFPs) are found in fishes living in both the Arctic and Antarctic polar ocean environments, where seawater temperatures are often as low as the freezing point of seawater (about -1.9 C). Any temperate fish species exposed to such extreme conditions would instantly freeze, as their equilibrium freezing point is near -0.7 C.10-12 Polar fishes that survive under these freezing temperatures have been found to produce a range of (glyco)proteins that have the capacity to lower the freezing point of their blood by inhibiting the growth of ice crystals that enter their bodies. Antifreeze (glyco)proteins are believed to operate by adsorbing onto specific faces of ice crystals, usually (but not exclusively) the prism faces, thereby *To whom correspondence should be addressed. Professor David E Williams, Department of Chemistry, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Telephone 64 9 373 7599 ext 89877. E-mail: [email protected]. Internet: www.che.auckland.ac.nz. pubs.acs.org/crystal

Published on Web 10/26/2010

inhibiting crystal growth, predominantly along the a-axis.9,13-15 This alters the growth habit of the ice crystal from a hexagonal plate, creating a hexagonal bipyramid in which the now stepped prism faces are assumed to be covered by a network of antifreeze (glyco)proteins.16 Once the crystal reaches this typical geometry, its growth is stopped until the temperature further decreases below a new depressed freezing point, also called the “hysteresis freezing point”, at which crystal growth accelerates. The difference between the melting point and the depressed freezing point is referred to as the thermal hysteresis. When the temperature decreases further below the depressed freezing point, the ice crystals grow quickly along the c-axis, giving rise to very fine hexagonal-shaped spicules, eventually with pyramidal tips.17,18 The expression of thermal hysteresis behavior during freezing point depression is implicit in the original definition of antifreeze activity.6,19 Antarctic fish AFGPs consist predominantly of a repetitive three amino acid unit (Ala-Ala-Thr)n with the disaccharide β-D-galactose-R-D-N-acetyl-galactosamine attached to the hydroxyl oxygen of each threonyl residue.19-21 Their molar masses vary between 2.6 and 33.7 kDa, which corresponds to 4-50 repetitions of the glycosylated tripeptide unit.16 These isoforms are conveniently grouped into eight size classes based on their electrophoretic properties, with AFGP1 representing the largest and AFGP8 the smallest of the notothenioid antifreeze glycoproteins.22 Some minor sequence variations have been identified in the AFGPs; Ala-Ala-Thr units, for example, are occasionally replaced by Pro-Ala-Thr in the r 2010 American Chemical Society

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Scheme 1. Synthesis of GalNAc-Thr Building Block 5

Scheme 2. Synthesis of Gal-Thr Building Block 8

smaller AFGPs.23-25 The conformation of the AFGPs in the presence of ice has not been elucidated although most researchers agree on an amphipathic structure with a hydrophilic face containing exposed hydroxyl groups of the sugars and a hydrophobic face containing the exposed methyl groups of both the amino acids and the N-acetylamine residues.26-38 The synthesis of AFGPs is a long and expensive process requiring a large number of steps.39-45 In order to find simpler compounds that exhibit antifreeze activity, numerous analogues of the AFGPs have been synthesized by various research groups.40,41,46-52 Notable among these studies, Tachibana et al.46 demonstrated that the terminal galactose of the native disaccharide is not essential for observing a thermal hysteresis, although the compounds substituted with N-acetyl-galactosamine monosaccharide were much less active than the native AFGP. In contrast to previous research, which has emphasized the study of thermal hysteresis or of recrystallization-inhibition,46,50,52-57 the objective of the present work is to explore the use of AFGP analogues to modify the growth habit of ice and to understand the factors that affect growth habit modification. Following Tachibana et al.46 but using solid-phase peptide synthesis in a method similar to that of Heggemann et al.,52 we synthesized a range of analogues derived from AFGP8 (14 residues) which have N-acetyl-galactosamine or galactose moieties attached to their threonines. We have also synthesized analogues in which the prolines have been substituted with alanines in the AFGP8 protein sequence. We demonstrate that the effects of the analogues on ice crystal growth habit are sensitively dependent on the nature of the sugar substituents, which we associate with a change in the solution conformation of the glycoprotein. The effect can also be greatly altered by a change in the chemical potential of the glycoprotein in the solution, achieved by the addition of other solutes. We associate growth habit modification with adsorption onto ice, demonstrate a simple NMR method for detecting this event, show a continuum of behavior connecting growth habit modification and thermal hysteresis, and rationalize

the behavior with an elementary kinetic model adapted from other studies where a kinetic description of adsorption is used to explain thermal hysteresis.58-63 Experimental Section Synthesis of Glycoprotein Analogues. The synthesis of Fmoc-RGalNAc-threonine 5 is summarized in Scheme 1. 3,4,6-Triacetyl-2azido-2-desoxygalactopyranosyl nitrate 1 was synthesized using a method similar to the one reported by Heggemann et al.52 Saccharide 1 was converted into 2 by displacement of the nitrate group by acetate. The key glycosylation of Fmoc-threonine-O-allyl by galactosyl donor 2 was then performed using the Lewis acid BF3 3 OEt2, and the two anomers were separated by column chromatography to yield R-glycoamino acid 3. Reductive acetylation of the azide group gave 4, which upon deprotection of the allyl ester provided the Fmoc-protected building block 5 in good yield. Readily available 1,2,3,4,6-penta-6-acetyl-D-galactopyranose 6 was used as the starting material for the synthesis of Fmoc-β-Galthreonine 8 (Scheme 2). Compound 6 was successfully coupled to Fmoc-threonine-O-allyl using the same method as described above to give 7. Deprotection of the allyl ester afforded the glycosylated amino acid 8. Solid phase peptide synthesis of the glycopeptides 11-13 (Table 1) using building blocks 5 and 8, as well as peptide 10, was carried out using an Fmoc/tBu strategy on a Liberty Microwave Peptide Synthesizer (CEM Corporation, Mathews, NC). Final purification was performed using reverse-phase HPLC. The glycoproteins were characterized by LC-MS. For more details about the synthesis and the characterization of the synthesized compounds, see the Supporting Information. NMR Kinetic Melting Experiment. 1H NMR spectra were recorded on a Bruker DRX spectrometer operating at a 1H frequency of 400 MHz. Chemical shifts (δ) were expressed in parts per million, and TMS served as an internal standard (δ = 0 ppm). The samples were dissolved in distilled water at similar molar concentration (1.75 10-2 mol 3 L-1) and then fitted into a 3 mm diameter NMR Norell-400 tube. The temperature of the NMR probe was calibrated using deuterated methanol following the method described by Findeisen et al.64 For flash-frozen samples, the sample tube was supercooled to -3 C and then plunged into liquid nitrogen and left to equilibrate at -3 C for 30 min. For slow-frozen sample preparation, the solution was flash-frozen and then slowly melted

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Table 1. Structure, Activity, and Melting NMR Experiment Results for Various Compounds and the Synthetic Analoguesa

a

NMR spectra are pictured on the same scale (see Supporting Information).

at room temperature until only a single tiny crystal remained in the tube. Slow freezing was then undertaken by leaving the tube in a bath at -3 C for 30 min. The sample tube was then fitted into a 5 mm diameter NMR Norell-400 tube containing CDCl3 previously

stabilized at -3 C. Samples were inserted into the instrument, which was equilibrated at -3 C and left for 10 min to allow temperature stabilization. Locking and shimming were performed using the external CDCl3 signal. A few one-dimensional (1D)

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Figure 1. Representative 1H NMR spectra of the water signal during the melting of native AFGP8 (14) (1.75 10-2 mol 3 L-1). The lowest trace (blue) represents the frozen state (t = 0 s), whereas the other traces correspond to the evolution of the water signal during melting of native AFGP8 (a) slow frozen or (b) flash frozen. For full spectral details see the Supporting Information. 1

H NMR experiments were run at this point to equilibrate the system. Once equilibration was reached, the temperature was set to 0.2 C to slowly melt the sample. Standard 1D 1H NMR measurements were subsequently acquired every 52 s to follow the melting of the sample. Automatic shimming on z1 and z2 shims was used as the sample melted. Full spectra are provided in the Supporting Information. Crystal Growth Morphology Observation. Direct observation of ice crystal growth was performed using a microscope equipped with a nanoliter freezing point osmometer (Otago Osmometers, Dunedin, NZ). The setup included a JCV TK-1281 Color Video Camera used to record the ice crystal images. The sample wells of the temperature-controlled osmometer stage were filled with oil (Cargille’s A) into which a small aliquot (∼20 nL) of the sample was injected using a micropipet attached to a Gilmont micrometer syringe. The sample solution was rapidly frozen and then slowly warmed until a single ice crystal remained. The temperature was then slowly and carefully decreased in order to observe the growth of the crystal. Observation of the crystal morphology and the lack or presence of a thermal hysteresis was noted and recorded on video. We define thermal hysteresis as the difference between the melting and freezing points of a solution when there is no observable ice crystal growth over at least 1 h. Calibration of the nanoliter freezing point osmometer was done using pure water and standard solutions of glucose and sodium chloride. For every salt study, controls were first done with the salt alone (at similar concentration) in order to be sure that no shaping of the crystal was induced by the salt itself (see Supporting Information). The pH for each tested sample was measured to be between 6 and 8, and therefore, no buffer was used except in the case of tricarballylic acid, where the solution (pH 2) was corrected to pH 7 using sodium hydroxide before the introduction of analogue 13. Circular Dichroism Analysis. CD analysis was carried out on an Applied Photophysics Pi-Star 180. All measurements were performed at room temperature in quartz cells of 1 cm path length. Spectra were obtained with a 1 nm band width, and 64 scans were combined to improve the signal-to-noise ratio. A baseline correction was made for each sample, and all spectra were recorded between 190 and 260 nm. The analogue concentration used was 4 μmol 3 L-1. Molar residual ellipticity (θ) was calculated in deg 3 cm2 3 dmol-1.

Results Demonstration of AFGP8/Analogues Adsorption onto Ice by an NMR Method. Observation of the water signal during the melting of pure ice showed a single peak at δ = 5.17 ppm for which the area of the signal increased as the ice melted. Observation of the water signal for a slow-frozen solution of N-acetyl-galactosamine (Table 1) or galactose (see Supporting Information) showed two resonances, one at δ = 5.17 ppm (which represents the signal for free water) and a second resonance at δ = 5.37 ppm or δ = 5.32 ppm (which re-

presents the signal for water interacting with N-acetylgalactosamine or galactose, respectively). During melting, both the free water and interactive peaks grew, but after 5-10 min only the free water peak grew (see Supporting Information). The slow freezing experiment performed with native AFGP8 showed only one resonance at δ = 5.17 ppm although the peak shape indicated that several similar environments of the water protons were present. The area of this peak increased during melting, conserving its original chemical shift (Figure 1a). In order to confirm that AFGP8 would normally interact with the water and shift its NMR signal if present in solution, the same experiment was repeated, but this time with a flash-frozen solution (in which AFGP8 molecules presumably do not have adequate time to adsorb onto the ice). This resulted in the detection of two resonances, one at δ = 5.17 ppm (pure water) and another at δ = 5.30 ppm, representing the water interacting with AFGP8 (Figure 1b). Analogues 10 and 11 at equivalent molar concentrations in the slow frozen state showed peaks at δ=5.48 ppm and δ = 6.16 ppm, respectively (Table 1), indicating the presence of compounds with strong hydrogen-bonding interactions with liquid water trapped in the ice. The free water resonance was absent in the frozen state but quickly appeared as melting began (see Supporting Information). The melting of a flash frozen solution of analogue 11 showed similar spectra with a resonance at δ = 5.15 ppm (which appears upon melting) and a resonance at δ = 5.50 ppm (see Supporting Information). Since the small resonances observed at δ = 5.00 ppm for 10 and δ = 5.10 ppm for 11 conserve the same area even upon complete melting, we attribute them to contamination of the CDCl3 with water. In the case of analogues 12 and 13, the flash-frozen experiments (see Supporting Information) showed two resonances for the water signal, one around δ = 5.15 ppm (free water) and one at δ = 5.40 ppm or δ = 5.26 ppm for 12 and 13, respectively (representing water interacting with the analogues). When slow frozen, the spectra were similar to that of the AFGP8 slow frozen sample (containing only one peak) somewhat broadened at δ=5.20 ppm and δ = 5.17 ppm for 12 and 13, respectively. As with the AFGP8 experiments (Figure 1b), these peaks (Table 1) conserved their chemical shift during melting. There were also distinct differences between the different compounds in the behavior of the NMR signals due to -CH3 protons (at 1-2.5 ppm) and -CH and -CH2 protons (in the range 3.5-5 ppm). These signals were detectable in all cases and were broad in the frozen solution but developed differently

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Figure 2. Representative 1H NMR spectra of the signal from the methyl protons during the melting of slow frozen solutions of (a) 10, (b) 11, (c) 12, (d) 13, and (e) 14 (recording terminated before melting was complete). All spectra were recorded at 1.75 10-2 mol 3 L-1.

Figure 3. CD spectra of synthetic analogues 10, 11, 12, 13, and natural AFGP8 14 at a concentration of 4 μmol 3 L-1 (water, room temperature).

as the frozen solution melted and the resonances sharpened. For galactose, N-acetyl-galactosamine, 10, and 11, the signals shifted significantly as the ice melted, by approximately -0.12, -0.17, -0.23, and -0.7 ppm, respectively. For 12, 13, and AFGP8, the signals retained the same chemical shift as the ice melted (Figure 2 and Supporting Information). Evaluation of Solution Conformation by Circular Dichroism. The CD spectra of the native AFGP8 (14), as well as analogues 10, 11, 12, and 13, are given in Figure 3. Native AFGP8

showed a maximum peak at 220 nm and a minimum around 190 nm. Analogues 12 and 13 showed spectra similar to those of the native AFGP8, with a positive band of the signal at 223 and 217 nm, respectively, and a negative band around 195 nm, with the signal for 12 being weaker than the signals for 13 and 14. Analogues 10 and 11 displayed minima around 195 nm but were without distinct maxima. Dependence of Crystal Growth Morphology on AFGP8/ Analogues Structure and Concentration. Three variables were

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explored: the sugar substituent, Gal-GalNAc, Gal, or GalNAc (analogues 11, 12, and 14); proline substitution with alanine (analogues 12 and 13); and the analogue concentration. The correlation of (glyco)protein composition, ice crystal shape, and NMR signal evolution on melting is summarized in Table 1. In accordance with the literature for small antifreeze glycoproteins,9 hexagonal bipyramidalshaped ice crystals with a thermal hysteresis of 0.35 C ((0.01 C, N = 2) were observed for native AFGP8 (14) at 20 mg 3 mL-1. When crystal growth morphology was determined in pure water, as well as in solutions of alanine, threonine, galactose, or N-acetyl-galactosamine, the slightest decrease in temperature induced single crystal growth equally in all directions (without faceting) until the whole sample was frozen. Similarly, no faceting was observed for solutions of analogue 10 at 10, 20, 40, and 80 mg 3 mL-1, while analogue 11 only displayed faceting at 80 mg 3 mL-1, at which concentration irregular hexagonal plates were observed without thermal hysteresis (Figure 4). The presence of analogues 12 and 13 in solution modified the growth habit of ice even at low concentrations, showing a concentrationdependence in the crystal shape they induced. At low concentrations (5-10 mg 3 mL-1) of analogues 12 and 13, thin hexagonal plates were observed (Figure 5). At higher concentrations (20-30 mg 3 mL-1), the crystals were hexagonal rods. It is noteworthy that the growth of the crystals was rather irregular, with some facets growing faster than others, resulting periodically in irregular hexagonal-shaped crystals. At 40 mg 3 mL-1, truncated hexagonal bipyramids were observed without thermal hysteresis. The kinetics of the crystal growth appeared to significantly slow down as the

Figure 4. Ice crystal morphology obtained in the presence of analogue 11 at (a) 10 mg 3 mL-1, (b) 20 mg 3 mL-1, and (c) 80 mg 3 mL-1.


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analogue concentration increased. At 80 mg 3 mL-1, hexagonal bipyramid-like crystals were observed, with a thermal hysteresis of 0.14 C ((0.1 C, N = 4) for 12 (see Supporting Information) and 0.15 C ((0.07 C, N=4) for 13 (Figure 5). When the temperature was further decreased below the hysteresis freezing point, the crystal still conserved its bipyramidal shape but grew at an extremely low rate. Synergistic Effects on the Crystal Growth Morphology of AFGP Analogues in Conjunction with Simple Solution Additives. The effects of various solution additives (trisodium citrate, tricarballylic acid, LiCl, NaCl, KCl, CsCl, Na2SO4, urea, and DMSO) have been investigated, with the salts being chosen so as to sample the Hofmeister series.65 Addition of urea, sodium sulfate, or DMSO did not affect the activity of analogue 13 (see Supporting Information). The addition of various chloride salts to a 10 mg 3 mL-1 solution of analogue 13 showed that the shape of the crystals obtained was significantly dependent on the size of the additive’s cation (Figure 6). Increasing the size of the cation had a similar effect on the crystal shape as increasing the concentration of the analogue. Addition of LiCl, for example, did not show any change in the way analogue 13 modified ice crystal growth, whereas CsCl gave rise to bipyramidal crystals with an observable but barely measurable thermal hysteresis of the order of 0.01 C ((0.01 C, N = 3). Again, the rate of crystal growth appeared to progressively decrease as the cation size increased. CsCl was also a potent promoter of the activity of the less-active analogues. Thus, in a similar way to analogue 13, addition of 1 mol 3 L-1 of CsCl to a 10 mg 3 mL-1 solution of analogue 11 (at which concentration the crystal habit was the same as water) caused the formation of hexagonal plate-shaped crystals (Figure 6e). Trisodium citrate was a particularly active promoter of the antifreeze activity of the analogues in terms of growth habit modification and thermal hysteresis. Addition of sodium citrate to a 10 mg 3 mL-1 solution of analogue 13 dramatically enhanced antifreeze activity, giving rise to bipyramidalshaped ice crystals with a thermal hysteresis of 0.36 C ((0.04 C, N = 4) (Figure 7a). Addition of sodium citrate to a solution (10 mg 3 mL-1) of analogue 11 surprisingly led to a thermal hysteresis of 0.90 C ((0.2 C, N = 3) and very small ice crystals (Figure 7b). Tricarballylic acid differs from

Figure 5. Ice crystal morphology obtained in the presence of analogue 13 at (a) 10 mg 3 mL-1, (b) 20 mg 3 mL-1, (c) 40 mg 3 mL-1, and (d) 80 mg 3 mL-1; thermal hysteresis = 0.15 C ((0.07 C, N = 4).

Figure 6. Ice crystal morphology obtained in the presence of (a) 10 mg 3 mL-1 of 13 and 1 mol 3 L-1 of LiCl, (b) 10 mg 3 mL-1 of 13 and 1 mol 3 L-1 of NaCl, (c) 10 mg 3 mL-1 of 13 and 1 mol 3 L-1 of KCl, (d) 10 mg 3 mL-1 of 13 and 1 mol 3 L-1 of CsCl, thermal hysteresis = 0.01 C ((0.01 C, N = 3), and (e) 10 mg 3 mL-1 of 11 and 1 mol 3 L-1 of CsCl.

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Figure 7. Ice crystal morphology obtained in the presence of 10 mg 3 mL-1 of (a) 13 with 1 mol 3 L-1 of trisodium citrate, thermal hysteresis = 0.36 C ((0.04 C, N = 4), (b) 11 with 1 mol 3 L-1 of trisodium citrate, thermal hysteresis = 0.90 C ((0.2 C, N = 3), and (c) 13 with 1 mol 3 L-1 of tricarballylic acid (pH = 7), thermal hysteresis = 0.27 C ((0.03 C, N = 2).

trisodium citrate in the lack of a hydroxyl group at position 3. A 1 mol 3 L-1 solution of tricarballylic acid (pH adjusted to 7) in conjunction with 10 mg 3 mL-1 of analogue 13 promoted the formation of tiny hexagonal bipyramidal ice crystals (Figure 7c) and induced a thermal hysteresis of 0.27 C ((0.03 C, N = 2). For these solutions of analogue 11 or 13 in the presence of either trisodium citrate or tricarballylic acid, an extremely low rate of crystal grow was observed once the temperature was further decreased below the hysteresis freezing point. Discussion NMR Evidence for Adsorption onto Ice and Interaction with Water in Ice Pockets. There is extensive literature on the NMR spectroscopy of frozen samples,66 discussing, among other matters, phase diagram determination,67,68 water in gels,69 hydration of biomolecules66 including food components,70 and alterations in hydrogen bonding as a consequence of confinement in small voids71 or by interaction with surfaces.72-74 The observation of reasonably sharp highresolution NMR water signals in the frozen state at -3 C for solutions of the studied compounds indicates that there is liquid water remaining in the preparation, presumably as tiny pockets of solution concentrated in solute (and hence with depressed freezing points) that are trapped within the ice during the freezing process.67 This liquid water would show an NMR signal that is altered by its interactions with the solute. Alterations in hydrogen-bonding, in particular, are expected to cause measurable changes in the chemical shift of the water signal. Resonances due to solute dissolved in the unfrozen water are also expected to be visible in the NMR spectra. These should be broadened by the effects of increased viscosity and low temperature. As the frozen solution melts, the resonances will sharpen and the effects of the interactions in the concentrated local environment should dissipate as the trapped solution pockets become diluted and incorporated into the bulk water. Although there may have been residual salts remaining in the preparations of the analogues and of AFGP8 after purification, we have assumed that the concentrations would be negligible and that the observed effects are therefore dominated by the AFGP8/ analogue component. The amount of liquid water present within the ice was not too different in the different samples studied, as indicated by the NMR signal intensity. However, the NMR experiments showed distinct differences between galactose, N-acetylgalactosamine, and analogues 10 and 11 on the one hand and AFGP8 and analogues 12 and 13 on the other. The first group of compounds showed a wide and distinctive range of

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water proton environments with resonances shifted downfield, indicative of an increase in the average number of hydrogen bonds per water molecule.73 They also showed significantly shifted resonances for the protons associated with the sugar and protein. When allowed to freeze slowly, the second group of compounds showed neither effect. We can develop an interpretation based on differing adsorption onto the surface of ice crystals and on differing interactions with liquid water in the unfrozen solution pockets. We argue that the portion of the water resonance shifted by hydrogen bonding indicates the amount of protein that is in solution in the unfrozen solution pockets. The amount of unfrozen water and hence the integrated NMR signal intensity would be dependent on the amount of protein in solution but would also be altered by different interactions between the solute and water, reflected in changes in the thermodynamic activity. The integrated NMR signal intensity cannot therefore be used as a reliable guide to the amount of protein in solution. In the case of AFGP8 and analogues 12 and 13, the amount of water with altered hydrogen-bonding interaction was significantly dependent on the freezing rate, whereas this was not the case for analogue 11, studied as a representative of the other group. A reasonable interpretation of our observations is that AFGP8 and analogues 12 and 13 adsorb onto ice, diminishing the amount present in solution in the pockets, but analogue 11 does not. If compounds do not adsorb when water is flashfrozen but do adsorb when ice is grown slowly from a small nucleus, then the adsorption rate must be relatively slow. The literature shows face-specific adsorption of the native antifreeze molecules: our experiment can thus be interpreted as showing that the removal of the compounds from solution is kinetically limited by the rate of appearance of the particular adsorbing crystal faces. This interpretation is consistent with the observed change of crystal habit from c-axial growth to a-axial growth when the rate of freezing of solutions of AFGP1-5 is increased beyond a certain value.75 The broad range of water proton environments observed from the unfrozen solution pockets in the case of galactose, N-acetyl-galactosamine, and analogues 10 and 11 indicates a wide range of hydrogen-bonding interactions with a time scale for exchange between them that is relatively slow compared to the NMR chemical shift time scale. That these effects are not seen in the case of AFGP8 and analogues 12 and 13 again argues that in these examples the concentration of the compounds in solution may be diminished by the effects of adsorption onto ice. We also argue from this observation that AFGP8 and analogues 12 and 13 adopt conformations in which there is not such a range of environments as for galactose, N-acetyl-galactosamine, and analogues 10 and 11, and that the time scale for exchange between them is faster. Similar arguments apply to the behavior of the protein and sugar proton resonances: in the case of galactose, N-acetyl-galactosamine, and analogues 10 and 11, if the solution concentration is higher, then the effects of a change in viscosity and homogeneous interaction of the analogue (along with a temperature effect) will broaden and shift the resonances. In the case of AFGP8 and analogues 12 and 13, such consequences of a higher concentration are not observed, arguing for their adsorption onto ice. Correlation of AFGP8/Analogues Structure, Adsorption onto Ice, and Crystal Shape. Table 1 shows the relationship between the structures of the AFGP8/analogues, the crystal shape that they induce, and whether or not they adsorb onto

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ice. At the tested concentrations, analogues 10 and 11 do not adsorb onto ice and do not induce any particular shape to a growing ice crystal. Analogues 12 and 13 and native AFGP8 14 adsorb onto ice and modify its normal growth habit, providing evidence that adsorption onto ice is part of the mechanism of AFGP ice crystal growth habit modification. Analogues 12 and 13 do not induce any observable thermal hysteresis at such concentrations, suggesting that while binding onto ice seems to be necessary for growth habit modification, it is not sufficient in itself to induce thermal hysteresis. The lack of activity of 10 indicates the necessity of the sugar groups in order to bind onto ice and modify the ice growth habit. Results for analogue 11 suggest an important role for the N-acetylamine group of the N-acetyl-galactosamine and/or the R-linkage of the N-acetyl-galactosamine. In the literature, Tachibana et al. showed that removing the N-acetylamine group of the N-acetyl-galactosamine induced a loss of thermal hysteresis as well as a loss of crystal shaping properties.46 In a different approach, we synthesized the β-linked galactose analogue. Analogue 11 does not exhibit any crystal shaping at concentrations under 80 mg 3 mL-1, and only slight shaping is observed at 80 mg 3 mL-1. Direct comparison between the results of Tachibana et al. and the effects of the analogues synthesized in this study is not possible due to the differences in length. However, both studies show that the N-acetylamine group is a key factor in enhancing the binding onto ice and subsequently in modifying the normal growth habit of ice. CD spectra (Figure 3) show a difference in conformation for analogue 11 compared to the analogues containing an N-acetylamine group (12-14). Mimura et al. pointed out the possibility of a hydrogen bond between the amide proton of N-acetylgalactosamine and the carbonyl oxygen of threonine, to which the GalNAc is covalently linked.26 It can be argued that this intramolecular interaction would serve to orientate the sugar moiety with respect to the protein backbone, thus stabilizing a conformation of the glycoprotein more favorable for binding onto ice. The role of proline residues also warrants some discussion. In the present study, replacing proline residues by alanine (13) did not induce any significant difference in the analogue’s behavior, for ice growth habit modification as well as thermal hysteresis, suggesting that such a substitution does not modify the conformation of the glycoprotein enough to affect its adsorption onto ice. These results are in agreement with those of Schrag et al.,76 who argued that substitution of the proline residues by alanine does not affect thermal hysteresis. CD Solution Conformation of AFGP8 and Its Analogues. The CD spectra observed for the native AFGP8 (14) can be related to different secondary structures such as a three-fold left-handed helical structure,77 a polyproline helix,78 or even a random coil structure,79 all of which have been proposed for the native AFGPs in the literature.30,31,35,36 Analogues 12 and 13 show spectra quite similar to that of the native AFGP8, indicating that elimination of the terminal galactose of AFGP8 does not affect the glycoprotein solution conformation significantly. The small differences observed between the spectra are probably due to the number of proline residues in the glycoproteins. Analogue 12 contains two proline residues; analogue 13 does not contain any, while native AFGP8 is a combination of various sequences mostly containing one or two prolines. The results show that as the


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number of proline residues increases in the sequence, the positive band of the signal is red-shifted and weakens. Published reports suggest a red-shift of the negative band as the proline content increases because of the contribution of the tertiary amide chromophores.52 Taking into account the work of Rath et al.,79 we argue that having more proline residues in the sequence will most likely favor a polyproline II conformation at the expense of a random coil, since the polyproline II signal is more red-shifted than a random coil signal. Different spectral features are observed in the case of analogues 10 and 11, implying a conformational difference between the glycoproteins that do not adsorb onto ice (10 and 11) and those that do (12, 13, and 14). Having an appropriate solution conformation thus appears to be important for the glycoprotein to bind onto ice, with icebinding being necessary but not sufficient in itself to induce thermal hysteresis. Such a conformation does not require the presence of the terminal galactose attached to the N-acetylgalactosamine in the native AFGP, and substitution of at least two prolines by alanines is tolerated. The results are also consistent with the postulate that hydrogen-bond formation from the N-acetyl-galactosamine to the protein backbone, as well as the R-linkage of the N-acetyl-galactosamine, are essential elements in maintaining the active conformation. Simple Salts Affect the Interaction of AFGP8/Analogues with Liquid Water and Hence Modulate Their Interaction with Ice. Reports in the literature indicate that the presence of certain salts enhances the activity of native antifreeze (glyco)proteins. According to Evans et al.80 and Amornwittawat et al.,81 increasing the size of the cation or the anion increases the thermal hysteresis value. In the literature, sodium citrate is the additive that induces the greatest increase in the thermal hysteresis activity of insect antifreeze proteins.82,83 One possible explanation is that added solution species could salt-out the proteins, promoting their adsorption onto ice.84 As suggested by others,81,84 an effect like this might follow the Hofmeister series, by analogy to the effects on protein adsorption onto surfaces, and would be modified substantially by the effects of temperature upon cooling to the freezing point.65,85 We have observed that this is true to a degree with AFGP8 and its analogues. Significant effects were observed for strong kosmotropes such as citrate and Csþ, but no effect was observed for other salts that might have been expected to alter hysteresis. To discuss the influences of various additives, we consider their effects on the analogues 11, 12, and 13 in solution and assume a kinetic model for the effects (discussed below). The Hofmeister series reflects the effects both of specific interactions of solutes with (glyco)proteins and of competing solvation interactions with water.86,87 Here we focus on the competing solvation interactions, and since one favored conformation of AFGP8 and its analogues would have a predominantly hydrophilic (sugar) side and a predominantly hydrophobic (methyl) side, we consider the effects on the surface energy of the different faces.88 A strong kosmotrope will have an important disrupting effect on the water-structure, favoring the solvation of the hydrophobic face of the glycoprotein in solution. This will have an effect on the adsorption rate, and any such effect will depend on the surface conformation of the glycoprotein, since adsorption is dependent not only on the interaction of one face of the glycoprotein with ice but also on the interaction of the other face with the water adjacent to the surface. If one considers the

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hydrophobic face of the glycoprotein as the face binding to the ice, then the addition of a “structure-breaking” solute which “salts in” the hydrophobic face of the glycoprotein would decrease the adsorption rate of the glycoprotein onto ice. On the other hand, a surface conformation that has the sugars adsorbed to the ice with the hydrophobic groups of the glycoprotein exposed to the solution will be favored by ions that induce “structure-breaking” of the water, such as citrate. In this study, sodium citrate dramatically increased the activity of analogues 11 and 13, not only promoting extreme crystal shaping but also creating a thermal hysteresis of 0.9 and 0.36 C, respectively. Similarly, trisodium tricarballylate added to analogue 13 resulted in a similar ice crystal shaping, with a thermal hysteresis of 0.27 C. Small differences in thermal hysteresis values can be attributed to the fact that the citrate ions disrupt the structure of water more than tricarballylate ions because of their structural differences. As a consequence, two factors must be taken into account in predicting the salt-induced binding enhancement of a glycoprotein: the size of the ion and its affinity with water. Ions that strongly disrupt the water structure, such as Csþ or citrate, act as enhancers of the effects on growth habit of the present synthetic analogues, clearly arguing for a conformation in which the sugar face of the glycoprotein is attached to the ice crystal and the hydrophobic face is exposed to the liquid water. The effects of solution additives are also important because they demonstrate that compounds that are “inactive”, in terms both of growth habit modification and of thermal hysteresis, can be made “active” just by the addition of a salt to the solution. There is a continuous hierarchy of effects: adsorption leads to growth habit modification, and increasing growth habit modification leads ultimately to a significant thermal hysteresis. Models for the Observed Behavior. A simple layer-by-layer, kink-step-ledge model, with reversible adsorption at kink sites on selected faces resulting in a change in the growth rate of that face dependent on the residence time of the adsorbate at the kink site, would account for the crystal shape changes in a reasonably straightforward way. However, such ideas are not consistent with concepts concerning the growth of ice, where the growth faces can be rough on a molecular scale—hence invalidating the idea of a kink site—and where the growth is significantly influenced by a “quasi-liquid” layer or transition zone at the crystal-fluid interface.89,90 The literature on antifreeze (glyco)proteins is dominated by a discussion of the thermal hysteresis, that is, in explaining why crystal growth is stopped. The continuation of growth with a sufficient undercooling is viewed as a nucleation problem.75,88 The models either have as the controlling parameter the curvature of a step edge, itself determined by the distance between pinning sites,91,92 or the line tension of a step.93 Significance of Adsorption Kinetics in Different Models. The mechanism by which the adsorption of the antifreeze molecules alters the nucleation condition remains a subject for debate. There is particular concern as to whether the adsorption of antifreeze (glyco)proteins is kinetically reversible or irreversible on the time scale of observations. Models59,60,94-96 based on a Langmuir isotherm for adsorption naturally assume kinetic reversibility, but these models have a weakness in that they do not specify any mechanism connecting the observed thermal hysteresis to the surface coverage, instead assuming a simple proportionality. They have been criticized75 because they do not have any built-in

Peltier et al.

mechanism by which growth on a particular face can be completely stopped: an effect which is characteristic of the antifreeze activity of the natural proteins. Models in which adsorption is kinetically irreversible are consistent with observation, in that etching of ice crystals reveals the antifreeze molecules adsorbed to specific faces.97 Extremely strong binding with essentially no exchange between surface-bound molecules and solution molecules has been unequivocally demonstrated for some antifreeze proteins.98 However, there is no evidence that the antifreeze molecules are engulfed within growing ice crystals99 other than being trapped at grain boundaries within a rapidly formed seed.98 Furthermore, models postulating an irreversible adsorption at first sight do not have a built-in mechanism for a concentration-dependence of the antifreeze activity, since by implication the surface coverage on a face whose growth has stopped will increase to full coverage given enough time, regardless of the solution concentration of the antifreeze.75 For the case of irreversible adsorption, a concentrationdependence of the antifreeze activity can be rationalized by considering the balance between adsorption rate (which is concentration dependent) and crystal growth rate (which is dependent on undercooling) should the crystal growth restart.75 Should the adsorption rate be sufficiently high, then any growth nucleus is inhibited. For the case of reversible adsorption, a model consistent with the observation that crystal growth can be stopped can be developed by consideration of the fluctuations on the ice-water interface driven by the competing effects of addition and removal of water molecules from the interface and adsorption and desorption of antifreeze molecules.100 A formal development of a dynamic model was given by Sander and Tkachenko:101 the growth rate fell to zero at a critical surface coverage of adsorbate. A visualization of the resultant fluctuations governing the crystal growth is provided by the work of Wathen et al.,100 who presented a simulation model in which the kinetics of adsorption, desorption, and addition of molecules to the growing crystal were explicitly included. In the presence of AFP (insect and fish AFPs were modeled), the growth of a crystal face was stopped because adsorbed antifreeze molecules caused pinning and consequent curvature of the growth steps. These pinned growth steps, however, were constantly bulging and shrinking. If a pinning molecule desorbed, then the growth step bulging around it did not necessarily spontaneously advance and could possibly shrink. The critical control parameters would be the surface density and residence time of antifreeze relative to the time scales for addition and desorption of water molecules to different types of growth site, classified by Wathen et al.100 as having 1, 2, or 3 hydrogen bonds to neighbors. In the current study, analogues 11, 12, and 13 present a concentration- and salt-dependence on the ice crystal shape that they induce, and a smooth transition to an observable thermal hysteresis with change of these variables: such results argue for the existence of a kinetically reversible interaction between the glycoproteins adsorbed onto the ice and those in solution. We note that there seem to be two different phenomena to consider. The first is the gradual and essentially continuous change of behavior from no effect through to strong growth modification and thermal hysteresis that we have described for our synthetic analogues, and the extreme behavior shown by the natural AFGP that has been graphically illustrated by Knight and DeVries.75 These

Article

authors showed that when the undercooling is sufficient to restart crystal growth, an explosive growth of completely different morphology occurs. The latter case can be considered as a limit of behavior in which the adsorption is extremely strong. Nature of the Adsorption Interaction. Another point concerns the nature of the adsorption interaction of the antifreeze molecule with ice: either specific site-dependent adsorption governed by matching of ice lattice spacings to the distance between surface-binding groups on the antifreeze in particular assumed adsorption conformations88,97,102 or an oriented adsorption in which the interaction with the interfacial “quasi-liquid” layer of water is a significant element.61,99 Zepeda et al.99 showed that very low concentrations of AFGP (5 μg 3 mL-1) changed the mode of growth of the ice crystal from a spherical form to a sharply faceted form characteristic of layer-by-layer growth. While the faces whose growth had stopped had apparently large amounts of AFGP adsorbed, control of the crystal growth seemed to be determined by a relatively small proportion, initially undetectable, of the adsorbed molecules, the remainder of which were relatively weakly bound and not necessarily effective in stopping the growth of a crystal plane. The implication of this work is that the interphase region, of water immediately adjacent to the ice crystal, is modified as a consequence of the initial strong adsorption such that further antifreeze is accumulated within it. This model invokes a distance-dependent structure of water in the interfacial layer, which is modified to match the solvation requirements around the antifreeze molecules, and derives much of its justification from molecular dynamics calculations of the ice-water interface structure.89,90 A common factor appears to be the possibility of an amphipathic conformation of the antifreeze molecule, in which one face is hydrophobic and another hydrophilic. An extreme example of such a conformation is provided by the “hyperactive” antifreeze protein from the snow flea.103 In the case of our analogues, we argue that the possibility of an intramolecular hydrogen bond between the amide proton of N-acetyl-galactosamine and the carbonyl oxygen of threonine26 favors a conformation with sugars on one face of the molecule and methyls on the other. The native disaccharides would have a greater coverage of sugar on the hydrophilic face of the molecule than would our monosaccharides. Such a conformation would favor hydrogen-bonding interactions with particular ice crystal faces. We argue that the adsorption of the antifreeze molecules is determined not only by the interaction of particular elements of the surface of the molecules with a specific ice crystal plane but also by the interaction of other elements of different parts of the molecular surface with the semistructured water immediately adjacent to the interface. The local structure is different adjacent to different crystal planes of ice and is no doubt also altered by other solutes present in the solution at sufficient concentration. A further subtlety could be that the dynamics of these interphase water molecules would be locally affected by the presence of the antifreeze, thus affecting the rate constants for addition and removal of water from the ice. The idea has some similarity to the model proposed by Kristiansen and Zachariassen.92 These authors suggested that antifreeze (glyco)proteins have two effects: the first is to “structure” the interfacial water to promote the emergence of specific crystal planes, and the second is to adsorb upon these planes and pin their growth. They argued that the effects are a subtle balance of hydrophobic and


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hydrophilic forces, where the hydrophobic forces control the formation of particular crystal planes and the hydrophilic forces determine attachment of the antifreeze to the emergent plane. Simple Kinetic Model for Ice Crystal Growth Habit Modification. A simple kinetic scheme is helpful for qualitative interpretation:

A step, S, can advance (curvature-dependent rate coefficient kg) or be pinned by a glycoprotein molecule (SA): ka represents the rate constant of adsorption and kd the rate constant of desorption. Writing the rate of the crystal growth normal to the step as v = kg[S] gives the speed of growth along a face of the ice crystal: v ¼ kg

kd ka ½A þ kd

Alteration of the rate of growth requires a sufficiently high adsorption rate constant, a sufficiently low desorption rate constant, and, depending on these values, a sufficiently high adsorbate concentration. The differences in activity observed for the various compounds are then due to a difference in the rate constant value. This equation also explains the slow kinetics of crystal growth when the concentration of each analogue ([A]) is increased, and it explains why a thermal hysteresis was only observed in the presence of a high concentration of 12 and 13, with this thermal hysteresis being due to crystal growth with a rate close to zero. The effect of antifreeze adsorption on crystal shape is due to the differences in the rate constants for the different types of surface sites (corresponding to the growth of different crystal planes). In order to explain the more complex distorted crystal geometries, the variation of concentration of adsorbate with time within the small pocket of sample, and the effects of developing concentration gradients of the adsorbate also need to be taken into account. As the crystal grows within the small pocket of sample, it consumes more and more water molecules and the concentration of the solutes in the solution surrounding the crystal increases. As a direct effect, the speed of growth of the faces where the analogues are bound will further decrease. To discuss the effects of various additives, we can draw on the simple idea of a linear free energy relationship between the equilibrium constant and the rate constants for a reaction. If the chemical potential of the glycoprotein in solution is decreased (increased), then the rate constant for adsorption will be decreased (increased) and that for desorption increased (decreased). The rate constants will thus be affected by the solvation of the glycoprotein in the bulk solution and on the surface. A solute that tended to increase the chemical potential of the glycoprotein in solution, such as the strong Hofmeister species citrate or Csþ, would tend to increase the adsorption rate of the glycoprotein, rationalizing the increase in activity observed in the presence of such additives.

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Conclusion New antifreeze glycoprotein analogues have been synthesized, and the ice crystal shapes that they induce have been characterized at different concentrations. A “kinetic melting experiment” tool based on 1H NMR allowed us to examine whether or not the compounds adsorb onto ice. The results show the importance of having an appropriate glycoprotein solution conformation, and they suggest that the N-acetylamine group and the R-linkage of the sugar are key features of this conformation. The absence of terminal galactose as well as substitution of at least some prolines by alanines does not seem to affect this conformation significantly. Observation of the ice crystal geometries obtained in the presence of the different analogues at different concentrations has shown that the terminal galactose, the N-acetylamine group, and the R-linkage act as enhancers of ice growth habit modification. The effects on the ice crystal shape induced by various additives clearly argue for a conformation of bound glycoprotein with the hydroxyls of the sugars facing the ice. An increase in the analogue concentration as well as of the addition of strong kosmotropes into the solution had the same effect on crystal shape. This effect could be related, using a simple kinetic model, to an increase in the adsorption rate of the analogues. As a consequence, we distinguish three ways of controlling ice crystal growth habit using AFGP analogues. The first approach is to affect the chemical potential of the AFGP analogue on the surface by tuning the analogue itself to make it more likely to adsorb onto the crystal surface. A second approach consists in simply modifying the concentration of these analogues. Finally, the last method of control is to modify the chemical potential of the analogue in the solution. Such modification can be done by the addition to the system of simple solution additives that do not adsorb onto the crystal but modify the interaction of the AFGP analogue with the solvent. A dependence of the effect of the solution additive on the size and the structure of the AFGP analogues was observed. These three approaches modulate the ice crystal growth habit by affecting the adsorption and desorption rate constants of the glycoproteins onto ice. Acknowledgment. We thank the Human Frontier Science Program, the MacDiarmid Institute for Advanced Materials and Nanotechnology, and the Maurice Wilkins Centre for Biodiscovery for financial support. We particularly thank Dr. Michael Schmitz for assistance with the NMR experiments. Supporting Information Available: Detailed synthesis and characterization of the building blocks, proteins, and glycoproteins; detailed structure of the analogues; kinetic melting NMR spectra; images of crystal growth experiments including blanks. This material is available free of charge via the Internet at http://pubs.acs.org.

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