Interactions of β-Helical Antifreeze Protein Mutants with Ice

The fold of the β-helical antifreeze protein from Tenebrio molitor (TmAFP) proved to be surprisingly tolerant of multiple amino acid substitutions, enabling the construction of a panel of mutants displaying grids of single amino acid types in place of the threonines on the ice-binding face. These mutants, maintaining the regularity of amino acid spacing found in the wild-type protein but with different functional groups on the surface, were tested for antifreeze activity by measuring thermal hysteresis and observing ice grown in their presence. We found that no mutant exhibited the dramatic activity of the wild-type version of this hyperactive antifreeze protein. However, mutants containing four valines or tyrosines in place of the threonines in the center of the TmAFP ice-binding face showed residual thermal hysteresis activity and had marked effects on ice crystal morphology. The results are discussed in the context of a two-stage model for the absorption−inhibition mechanism of antifreeze protein binding to ice surfaces

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

Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics

Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 18C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH

Universality of Persistence Exponents in Two-Dimensional Ostwald Ripening

We measured persistence exponents θ ( ϕ ) of Ostwald ripening in two dimensions, as a function of the area fraction ϕ occupied by coarsening domains. The values of θ ( ϕ ) in two systems, succinonitrile and brine, quenched to their liquid-solid coexistence region, compare well with one another, providing compelling evidence for the universality of the one-parameter family of exponents. For small ϕ , θ ( ϕ ) ≃ 0.39 ϕ , as predicted by a model that assumes no correlations between evolving domains. These constitute the first measurements of persistence exponents in the case of phase transitions with a conserved order parameter

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

Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas.

Bissoyi A, Reicher N, Chasnitsky M, et al. Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas. Biomolecules. 2019;9(10): 532.Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size

Freezing and Melting Hysteresis Measurements in Solutions of Hyperactive Antifreeze Protein from an Antarctic Bacteria

Antifreeze proteins (AFPs) evolved in cold-adapted organisms and serve to protect them against freezing in cold conditions by arresting ice crystal growth. Recently, we have shown quantitatively that adsorption of AFPs not only prevents ice from growing but also from melting. This melting inhibition by AFPs, which results in superheated ice (Celik et al, PNAS 2010), is not a well-known phenomenon. Here we present our recent findings in which the Ca2+-dependent hyperactive AFP from Marinomonas primoryensis (MpAFP) clearly displays this property. Additionally, we found that an ice crystal that is initially stabilized and protected by this type of AFP can be overgrown and then melted back to the original crystal. This repeatable process is likely due to melting inhibition, and supports the idea that AFPs bind irreversibly to ice surfaces