Antifreeze proteins modify the freezing process in planta

During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 degrees C to 1.2 degrees C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.     

Solution Structure of an Antifreeze Protein CfAFP-501 from <Emphasis Type="Italic">Choristoneura fumiferana</Emphasis>

Antifreeze proteins (AFPs) are widely employed by various organisms as part of their overwintering survival strategy. AFPs have the unique ability to suppress the freezing point of aqueous solution and inhibit ice recrystallization through binding to the ice seed crystals and restricting their growth. The solution structure of CfAFP-501 from spruce budworm has been determined by NMR spectroscopy. Our result demonstrates that CfAFP-501 retains its rigid and highly regular structure in solution. Overall, the solution structure is similar to the crystal structure except the N- and C-terminal regions. NMR spin-relaxation experiments further indicate the overall rigidity of the protein and identify a collection of residues with greater flexibilities. Furthermore, Pro91 shows a cis conformation in solution instead of the trans conformation determined in the crystal structure.Structural data have been deposited at PDB (1Z2F) and Chemical shift data at BMRB (bmrb 6111).     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

A theoretical model of a plant antifreeze protein from Lolium perenne.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein's inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.     

Lessons from nature for preservation of mammalian cells, tissues, and organs

The study of mechanisms by which animals tolerate environmental extremes may provide strategies for preservation of living mammalian materials. Animals employ a variety of compounds to enhance their survival, including production of disaccharides, glycerol, and antifreeze compounds. The cryoprotectant glycerol was discovered before its role in amphibian survival. In the last decade, trehalose has made an impact on freezing and drying methods for mammalian cells. Investigation of disaccharides was stimulated by the variety of organisms that tolerate dehydration stress by accumulation of disaccharides. Several methods have been developed for the loading of trehalose into mammalian cells, including inducing membrane lipid-phase transitions, genetically engineered pores, endocytosis, and prolonged cell culture with trehalose. In contrast, the many antifreeze proteins (AFPs) identified in a variety of organisms have had little impact. The first AFPs to be discovered were found in cold water fish; their AFPs have not found a medical application. Insect AFPs function by similar mechanisms, but they are more active and recombinant AFPs may offer the best opportunity for success in medical applications. For example, in contrast to fish AFPs, transgenic organisms expressing insect AFPs exhibit reduced ice nucleation. However, we must remember that nature's survival strategies may include production of AFPs, antifreeze glycolipids, ice nucleators, polyols, disaccharides, depletion of ice nucleators, and partial desiccation in synchrony with the onset of winter. We anticipate that it is only by combining several natural low temperature survival strategies that the full potential benefits for mammalian cell survival and medical applications can be achieved.     

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.     

Antifreeze proteins and their potential use in frozen foods

Antifreeze proteins (AFPs) are proteins that have the ability to modify the growth of ice, resulting in the stabilization of ice crystals over a defined temperature range and in the inhibition of the recrystallization of ice. AFPs are found in a wide range of organisms, including bacteria, fungi, plants, invertebrates and fish. Moreover, multiple forms of AFPs are synthesized within each organism. As a result, it should be possible to select an AFP with appropriate characteristics and a suitable level of activity for a particular food product. Antifreeze proteins may improve the quality of foods that are eaten while frozen by inhibiting recrystallization and maintaining a smooth texture. In foods that are frozen only for preservation, AFPs may inhibit recrystallization during freezing, storage, transport and thawing, thus preserving food texture by reducing cellular damage and also minimizing the loss of nutrients by reducing drip. Antifreeze proteins are naturally present in many foods consumed as part of the human diet. However, AFPs may be introduced into other food products either by physical processes, such as mixing and soaking, or by gene transfer.     

A hyperactive, Ca2+-dependent antifreeze protein in an Antarctic bacterium

In cold climates, some plants and bacteria that cannot avoid freezing use antifreeze proteins (AFPs) to lessen the destructive effects of ice recrystallization. These AFPs have weak freezing point depression activity, perhaps to avoid sudden, uncontrolled growth of ice. Here, we report on an uncharacteristically powerful bacterial AFP found in an Antarctic strain of the bacterium, Marinomonas primoryensis. It is Ca(2+)-dependent, shows evidence of cooperativity, and can produce over 2 degrees C of freezing point depression. Unlike most AFPs, it does not produce obvious crystal faceting during thermal hysteresis. This AFP might be capable of imparting freezing avoidance to M. primoryensis in ice-covered Antarctic lakes. A hyperactive bacterial AFP has not previously been reported.     

A comprehensive evaluation of the effects and mechanisms of antifreeze proteins during low-temperature preservation

During the past 10 years, it has become clear that the effects of antifreeze proteins (AFPs) on cell viability and on thermodynamic properties during low-temperature preservation are complex, even controversial. In this paper, these studies are reviewed systematically and some conclusions are drawn. It is shown that AFPs can display both protective and cytotoxic actions and both nucleation of ice and inhibition of ice crystal growth, depending on several factors; these include the specific storage protocol, the dose and type of AFP, the composition and concentration of cryoprotectant, and the features of the biological material. A novel model, incorporating some recent findings concerning these proteins, is proposed to explain this dual effect of AFPs during cryopreservation. AFP-ice complexes have some affinity interactions with cell membranes and with many other molecules present in cryopreservation solutions. When the intensity of these interactions reaches a certain level, the AFP-ice complexes may be induced to aggregate, thereby inducing ice nucleation and loss of the ability to inhibit recrystallization.     

Peptide backbone circularization enhances antifreeze protein thermostability

Antifreeze proteins (AFPs) are a class of ice‐binding proteins that promote survival of a variety of cold‐adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large‐scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein‐mediated N‐ and C‐terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20‐Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice‐binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice‐affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild‐type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs.     

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.     

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9 °C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.     

Direct visualization of spruce budworm antifreeze protein interacting with ice crystals: basal plane affinity confers hyperactivity

Antifreeze proteins (AFPs) protect certain organisms from freezing by adhering to ice crystals, thereby preventing their growth. All AFPs depress the nonequilibrium freezing temperature below the melting point; however AFPs from overwintering insects, such as the spruce budworm (sbw) are 10-100 times more effective than most fish AFPs. It has been proposed that the exceptional activity of these AFPs depends on their ability to prevent ice growth at the basal plane. To test the hypothesis that the hyperactivity of sbwAFP results from direct affinity to the basal plane, we fluorescently tagged sbwAFP and visualized it on the surface of ice crystals using fluorescence microscopy. SbwAFP accumulated at the six prism plane corners and the two basal planes of hexagonal ice crystals. In contrast, fluorescently tagged fish type III AFP did not adhere to the basal planes of a single-crystal ice hemisphere. When ice crystals were grown in the presence of a mixture of type III AFP and sbwAFP, a hybrid crystal shape was produced with sbwAFP bound to the basal planes of truncated bipyramidal crystals. These observations are consistent with the blockage of c-axial growth of ice as a result of direct interaction of sbwAFP with the basal planes.     

The basis for hyperactivity of antifreeze proteins

Antifreeze proteins (AFPs) bind to the surface of ice crystals and lower the non-equilibrium freezing temperature of the icy solution below its melting point. We have recently reported the discovery of three novel hyperactive AFPs from a bacterium, a primitive insect and a fish, which, like two hyperactive AFPs previously recognized in beetles and moths, are considerably better at depressing the freezing point than most fish AFPs. When cooled below the non-equilibrium freezing temperature, ice crystals formed in the presence of any of five distinct, moderately active fish AFPs grow suddenly along the c-axis. Ice crystals formed in the presence of any of the five evolutionarily and structurally distinct hyperactive AFPs remain stable to lower temperatures, and then grow explosively in a direction normal to the c-axis when cooled below the freezing temperature. We argue that this one consistent distinction in the behaviour of these two classes of AFPs is the key to hyperactivity. Whereas both AFP classes bind irreversibly to ice, the hyperactive AFPs are better at preventing ice growth out of the basal planes.     

Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

利用伪氨基酸组分和支持向量机预测抗冻蛋白

抗冻蛋白是一类具有提高生物抗冻能力的蛋白质。抗冻蛋白能够特异性的与冰晶相结合,进而阻止体液内冰核的形成与生长。因此,对抗冻蛋白的生物信息学研究对生物工程发展。提高作物抗冻性有重要的推动作用。本文采用由400条抗冻蛋白序列和400条非抗冻蛋白序列构成数据集,以伪氨基酸组分为特征,利用支持向量机分类算法预测抗冻蛋白,对训练集预测精度达到91．3％,对测试集预测精度达到78．8％。该结果证明伪氨基酸组分能够很好的反映抗冻蛋白特性,并能够用于预测抗冻蛋白。     

Devitrification and recrystallization of nanoparticle-containing glycerol and PEG-600 solutions

Nanoparticles in solution offer unique electrical, mechanical and thermal properties due to their physical presence and interaction with the state of dispersion. This work is aimed to study the effects of hydroxyapatite (HA) nanoparticles on the devitrification and recrystallization events of two important cryoprotective solutions used in cell and tissue preservation namely glycerol (60% w/w) and PEG-600 (50% w/w). HA nanoparticles (20, 40 or 60 nm) were incorporated into solutions at the content of 0.1% or 0.5% (w/w), and were studied by differential scanning calorimeter (DSC) and cryomicroscopy. The presence of nanoparticles does not change the glass transition temperatures and melting temperatures of quenched solutions, but significantly affects the behavior of devitrification and recrystallization upon warming. Cryomicroscopic investigation showed the complex interactions among solution type, nanoparticle size and nanoparticle content, which apparently influence ice crystal growth or recrystallization in the quenched dispersions. These findings have significant implications for biomaterial cryopreservation, cryosurgery, and food manufacturing. The complexity of ice crystal growth kinetics in nanoparticle-containing dispersions remains to be poorly understood at the moment.