Modeling the Influence of Antifreeze Proteins on Three-Dimensional Ice Crystal Melt Shapes using a Geometric Approach

The melting of pure axisymmetric ice crystals has been described previously by us within the framework of so-called geometric crystal growth. Nonequilibrium ice crystal shapes evolving in the presence of hyperactive antifreeze proteins (hypAFPs) are experimentally observed to assume ellipsoidal geometries ("lemon" or "rice" shapes). To analyze such shapes we harness the underlying symmetry of hexagonal ice Ih and extend two-dimensional geometric models to three-dimensions to reproduce the experimental dissolution process. The geometrical model developed will be useful as a quantitative test of the mechanisms of interaction between hypAFPs and ice.Comment: 15 pages, 5 figures; Proc. R. Soc. A, Published online before print June 27, 201

New insights into ice growth and melting modifications by antifreeze proteins

Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs

Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules

Cryo-protective effect of an ice-binding protein derived from Antarctic bacteria

Cold environments are populated by organisms able to contravene deleterious effects of low temperature by diverse adaptive strategies, including the production of ice binding proteins (IBPs) that inhibit the growth of ice crystals inside and outside cells. We describe the properties of such a protein (EfcIBP) identified in the metagenome of an Antarctic biological consortium composed of the ciliate Euplotes focardii and psychrophilic non-cultured bacteria. Recombinant EfcIBP can resist freezing without any conformational damage and is moderately heat stable, with a midpoint temperature of 66.4 °C. Tested for its effects on ice, EfcIBP shows an unusual combination of properties not reported in other bacterial IBPs. First, it is one of the best-performing IBPs described to date in the inhibition of ice recrystallization, with effective concentrations in the nanomolar range. Moreover, EfcIBP has thermal hysteresis activity (0.53 °C at 50 μm) and it can stop a crystal from growing when held at a constant temperature within the thermal hysteresis gap. EfcIBP protects purified proteins and bacterial cells from freezing damage when exposed to challenging temperatures. EfcIBP also possesses a potential N-terminal signal sequence for protein transport and a DUF3494 domain that is common to secreted IBPs. These features lead us to hypothesize that the protein is either anchored at the outer cell surface or concentrated around cells to provide survival advantage to the whole cell consortium

Inhibition of ice growth and recrystallization by zirconium acetate and zirconium acetate hydroxide.

The control over ice crystal growth, melting, and shaping is important in a variety of fields, including cell and food preservation and ice templating for the production of composite materials. Control over ice growth remains a challenge in industry, and the demand for new cryoprotectants is high. Naturally occurring cryoprotectants, such as antifreeze proteins (AFPs), present one solution for modulating ice crystal growth; however, the production of AFPs is expensive and inefficient. These obstacles can be overcome by identifying synthetic substitutes with similar AFP properties. Zirconium acetate (ZRA) was recently found to induce the formation of hexagonal cavities in materials prepared by ice templating. Here, we continue this line of study and examine the effects of ZRA and a related compound, zirconium acetate hydroxide (ZRAH), on ice growth, shaping, and recrystallization. We found that the growth rate of ice crystals was significantly reduced in the presence of ZRA and ZRAH, and that solutions containing these compounds display a small degree of thermal hysteresis, depending on the solution pH. The compounds were found to inhibit recrystallization in a manner similar to that observed in the presence of AFPs. The favorable properties of ZRA and ZRAH suggest tremendous potential utility in industrial applications

Ice recrystallization inhibition by ZRA and ZRAH.

<p>LC-PolScope images for (A) ZRA at pH 4.2, (B) ZRAH at pH 4.2, (C) 100 mM sodium acetate pH 4.2 (negative control), and (D) AFP III (40 µM solution). The colors represent the optical axis orientations of the ice which was determined by the Abrio birefringence detection system. The color map in the bottom right corner corresponds to the respective birefringence orientation. The images in the first and second columns show the initial frozen state at 4× and at 50× magnifications, respectively. Images in the last three columns were collected at an annealing temperature of –3°C, at times 0, 10, and 60 minutes. Note the grain boundaries that appeared during annealing only in the buffer sample. These boundaries shifts indicate recrystallization process.</p

Comparison of the effects of ZRAH and ZRA on the ice growth rates along the <i>c</i>-axis.

<p>The X-axis represents the temperature below T_m. The Y-axis represents the rate of growth of the radius of a seed ice crystal 20 µm in length. For ZRAH solutions, growth at ΔT >0.1°C could not be measured due to crystal orientation.</p

Ice crystal shaping during growth in the presence of (A) 100 mM sodium acetate (pH 4.2), (B, D, F) ZRAH (30 g/L) and (C, E, G) ZRA (13.3 g/L) solutions.

<p>Ice crystal shaping during growth in the presence of (A) 100 mM sodium acetate (pH 4.2), (B, D, F) ZRAH (30 g/L) and (C, E, G) ZRA (13.3 g/L) solutions.</p

The effect of ZRA and ZRAH on the growth rate of ice crystals along the <i>a</i>-axis.

<p>The X-axis represents the temperature below T_m. The Y-axis represents the rate of growth of the radius of the ice crystal. Each point represents the average of at least 3 measurements. (B) A magnified view of the area enclosed by the black rectangle in A. The horizontal dash-dot line indicates a growth rate of 0.02 µm/sec, which is the threshold value used to define the TH gap. The arrows indicate the semi-TH activity according to this modified definition. Note that according to the old threshold definition (0.2 µm/sec) the TH activity in the <i>a</i>-axis direction would be >0. 1°C.</p