A Ca2+-dependent bacterial antifreeze protein domain has a novel beta-helical ice-binding fold

AFPs (antifreeze proteins) are produced by many organisms that inhabit ice-laden environments. They facilitate survival at sub-zero temperatures by binding to, and inhibiting, the growth of ice crystals in solution. The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large(>1 MDa) hyperactive Ca2+-dependent AFP. We have cloned,expressed and characterized a 322-amino-acid region of the protein where the antifreeze activity is localized that shows similarity to the RTX (repeats-in-toxin) family of proteins. The recombinant protein requires Ca2+ for structure and activity, and it is capable of depressing the freezing point of a solution in excess of 2 degrees C at a concentration of 0.5 mg/ml, therefore classifying it as a hyperactive AFP. We have developed a homology-guided model of the antifreeze region based partly on the Ca2+-bound beta-roll from alkaline protease. The model has identified both a novel beta-helical fold and an ice-binding site. The interior of the beta-helix contains a single row of bound Ca2+ ions down one side of the structure and a hydrophobic core down the opposite side. The ice binding surface consists of parallel repetitive arrays of threonine and aspartic acid/asparagine residues located down the Ca2+-bound side of the structure. The model was tested and validated by site-directed mutagenesis. It explains the Ca2+-dependency of the region, as well its hyperactive antifreeze activity. This is the first bacterial AFP to be structurally characterized and is one of only five hyperactive AFPs identified to date.AFPS     

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.     

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.     

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.     

Functional Diversification and Evolution of Antifreeze Proteins in the Antarctic Fish Lycodichthys dearborni

Antifreeze proteins (AFPs) have independently evolved in many organisms. AFPs act by binding to ice crystals, effectively lowering the freezing point. AFPs are often at high copy number in a genome and diversity exists between copies. Type III antifreeze proteins are found in Arctic and Antarctic eel pouts, and have previously been shown to evolve under positive selection. Here we combine molecular and proteomic techniques to understand the molecular evolution and diversity of Type III antifreeze proteins in a single individual Antarctic fish Lycodichthys dearborni. Our expressed sequence tag (EST) screen reveals that at least seven different AFP variants are transcribed, which are ultimately translated into five different protein isoforms. The isoforms have identical 66 base pair signal sequences and different numbers of subsequent ice-binding domains followed by a stop codon. Isoforms with one ice-binding unit (monomer), two units (dimer), and multiple units (multimer) were present in the EST library. We identify a previously uncharacterized protein dimer, providing further evidence that there is diversity between Type III AFP isoforms, perhaps driven by positive selection for greater thermal hysteresis. Proteomic analysis confirms that several of these isoforms are translated and present in the liver. Our molecular evolution study shows that paralogs have diverged under positive selection. We hypothesize that antifreeze protein diversity is an important contributor to depressing the serum freezing point.     

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.     

Annealing condition influences thermal hysteresis of fungal type ice-binding proteins

The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis. In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c-axis of ice crystal. This burst pattern is similar to TisAFP and other hyperactive AFPs. The thermal hysteresis of NagIBP and TisAFP could be increased by decreasing a cooling rate to allow more time for the proteins to bind ice. This suggests the possible second binding of proteins occurs on the ice surface, which might increase the hysteresises to a sufficient level to prevent freezing of the brine pockets which habitat of N. glaciei. The secondary ice binding was described as that after AFP molecules bind onto the flat ice plane irreversibly, which was based on adsorption–inhibition mechanism model at the ice–water interface, convex ice front was formed and overgrew during normal TH measurement (no annealing) until uncontrolled growth at the nonequilibrium freezing point. The results suggested that NagIBP is a hyperactive AFP that is expressed for freezing avoidance.     

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.     

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.     

Structure and evolutionary origin of Ca(2+)-dependent herring type II antifreeze protein

In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca(2+)-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+)-dependent) lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+) and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+)-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+) through the coordination with a water molecule of the ice lattice. This Ca(2+)-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.     

Short Communication: Increased gene dosage augments antifreeze protein levels in transgenic Drosophila melanogaster

One of the principal environmental adaptations of certain fishes inhabiting polar and northern coastal waters is the synthesis of antifreeze proteins (AFPs). AFPs bind to and prevent the growth of nascent ice crystals, thus depressing the serum freezing point. The transgenic expression of AFP holds great promise for conferring freeze resistance to commercially important plant and animal species. Since fish at the greatest risk of freezing have multiple AFP gene copies in order to synthesize higher levels of this protein, we have evaluated this evolutionary strategy as a way to maximize AFP expression in a model transgenic host, the fruit fly Drosophila melanogaster. A construct in which AFP genes of the Atlantic wolffish are fused to the Drosophila yolk protein 1,2 promoter/enhancer region was transferred to flies through P-element mediated transformation. Several independent transgenic fly lines were used in genetic crosses to obtain multi-insert lines. Haemolymph freezing point depression (thermal hysteresis) was greater in homozygotes relative to heterozygotes for a given insert. Similarly, multi-insert lines consistently displayed greater haemolymph AFP activity than the single insert lines from which they were derived. The thermal hysteresis value obtained with a fly line harboring 8 AFP gene copies, 0.43 °C, represents the highest such value to date recorded in a transgenic host, and is even higher than the levels found in some AFP-producing fish.     

A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants.     

Two domains of RD3 antifreeze protein diffuse independently

Antifreeze proteins (AFPs) make up a class of structurally diverse proteins that help to protect many organisms from freezing temperatures by inhibiting ice crystal growth at temperatures below the colligative freezing point. AFPs are typically small proteins with a relatively flat, slightly hydrophobic binding region that matches the lattice structure of a specific ice crystal plane. The only known two-domain AFP is RD3 from the Antarctic eel pout. It consists of two nearly identical type III domains connected by a nine-residue linker. This protein exhibits higher activity than the single-domain protein at low concentrations. The initial solution structure of RD3 revealed that the domains were aligned so that the binding regions were nearly coplanar, effectively doubling the surface area for binding. A more recent report suggests that the domains may not be aligned in solution but rather diffuse independently. To resolve the issue, we have measured the NMR residual dipolar couplings using alignment media of stretched gels and filamentous phage to determine the relative orientation of the domains. We find that the two domains of RD3 are free to move relative to each other, within the constraint of the flexible nine-residue linker. Our data show that there is no strongly preferred alignment in solution. Furthermore, the flexibility and length of the linker are sufficient to allow the two domains to have their binding faces in the same orientation and coplanar for simultaneous binding to an ice crystal surface.     

鱼类抗冻蛋白的研究进展

抗冻蛋白 (AFP)可非依数性地降低溶液冰点 ,对冷冻细胞和胚胎具有高效的保护作用。目前的研究表明 ,不同的鱼类抗冻蛋白尽管都具有降低冰点的活性 ,但在结构和组成上又存在有较大的差异。根据其结构和化学组成 ,一般将它们分为 4大类 :AFP I、AFP II、AFP III和AFP IV。抗冻蛋白的编码基因为基因组中多拷贝基因家族的成员 ,其基因表达在很大程度上要受到季节变化的影响。目前 ,普遍使用吸附抑制假说来解释AFP非依数性降低溶液冰点的分子机制 ,但不同类抗冻蛋白在降低溶液冰点时的作用模式却不尽相同。现就鱼类的 4类抗冻蛋白的结构组成、基因性质、抗冻机制及其在细胞和胚胎冻存中的作用等领域的研究进展进行概括性综述     

Positive Darwinian Selection Promotes Heterogeneity Among Members of the Antifreeze Protein Multigene Family

A variety of organisms have independently evolved proteins exhibiting antifreeze activity that allows survival at subfreezing temperatures. The antifreeze proteins (AFPs) bind ice nuclei and depress the freezing point by a noncolligative absorption–inhibition mechanism. Many organisms have a heterogeneous suite of AFPs with variation in primary sequence between paralogous loci. Here, we demonstrate that the diversification of the AFP paralogues is promoted by positive Darwinian selection in two independently evolved AFPs from fish and beetle. First, we demonstrate an elevated rate of nonsynonymous substitutions compared to synonymous substitutions in the mature protein coding region. Second, we perform phylogeny-based tests of selection to demonstrate a subset of codons is subjected to positive selection. When mapped onto the three-dimensional structure of the fish antifreeze type III antifreeze structure, these codons correspond to amino acid positions that surround but do not interrupt the putative ice-binding surface. The selective agent may be related to efficient binding to diverse ice surfaces or some other aspect of AFP function. Received: 27 February 2001 / Accepted: 12 September 2001     

Hyperactive antifreeze protein from fish contains multiple ice-binding sites

Antifreeze proteins (AFPs) are produced to prevent freezing in many fish species that are exposed to icy seawater. There are a number of nonhomologous types of AFPs, diverse in both sequence and structure, which share the function of binding to ice and inhibiting its growth. We recently discovered a hyperactive AFP in the winter flounder and related species that is many-fold more active than other fish AFPs. Like the 3-4-kDa type I AFPs, it is alanine-rich and highly helical, but this 17-kDa protein is considerably larger and forms a dimer. We have sequenced the cDNA encoding this new AFP to gain insight into its structure and evolutionary relationship to the type I AFP family. The gene is clearly homologous to the righteye flounder type I AFP genes. Thus we have designated this protein "hyperactive type I AFP" (hyp-type I). The sequence of hyp-type I AFP supports a structural model in which two extended 195-amino acid alpha-helices form an amphipathic homodimer with a series of linked Ala- and Thr-rich patches on the surface of the dimer, each of which resembles ice-binding sites of type I AFPs. The superior activity of hyp-type I AFP may derive from the large combined surface area of the ice-binding sites, recognition of multiple planes of ice, and protection of the basal plane from ice growth.     

Conjugation of type I antifreeze protein to polyallylamine increases thermal hysteresis activity

Antifreeze proteins (AFPs) are ice binding proteins found in some plants, insects, and Antarctic fish allowing them to survive at subzero temperatures by inhibiting ice crystal growth. The interaction of AFPs with ice crystals results in a difference between the freezing and melting temperatures, termed thermal hysteresis, which is the most common measure of AFP activity. Creating antifreeze protein constructs that reduce the concentration of protein needed to observe thermal hysteresis activities would be beneficial for diverse applications including cold storage of cells or tissues, ice slurries used in refrigeration systems, and food storage. We demonstrate that conjugating multiple type I AFPs to a polyallylamine chain increases thermal hysteresis activity compared to the original protein. The reaction product is approximately twice as active when compared to the same concentration of free proteins, yielding 0.5 °C thermal hysteresis activity at 0.3 mM protein concentration. More impressively, the amount of protein required to achieve a thermal hysteresis of 0.3 °C is about 100 times lower when conjugated to the polymer (3 μM) compared to free protein (300 μM). Ice crystal morphologies observed in the presence of the reaction product are comparable to those of the protein used in the conjugation reaction.     

Polycarboxylates enhance beetle antifreeze protein activity

Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine-N,N,N',N',N',N'-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn(2+) coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed.     

Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast

Certain freeze-intolerant insects produce antifreeze proteins (AFPs) during overwintering including the spruce budworm (Choristoneura fumiferana) and yellow mealworm (Tenebrio molitor) AFP gene families. However, only a few of the isoforms, encoded by their multiple-copy gene families, have been characterized. When expressed in bacterial systems the insect AFPs have to be denatured and refolded in vitro, a procedure that is not uniformly successful, presumably due to the beta-helix structure and the requirement for disulfide bonds. In an attempt to overcome these difficulties, bacterial vectors and hosts that have been developed to produce soluble, folded proteins, as well as a yeast expression system (Pichia pastoris) were employed. Bacterial expression resulted in low quantities of active recombinant protein for certain isoforms. In contrast, both small and large-scale fermentation of recombinant AFP in Pichia yielded substantial protein production (100 mg/L) but functional ice binding activity of protein produced in three different transformed yeast strains (KM71, X33 or GS115) was low. Inappropriate O-linked glycosylation of the Thr-rich AFPs appeared to be partially reversed by mild chemical deglycosylation, but activity remained low. Substantial quantities, as well as activity were recovered when a fish AFP, with disulfide bonds, but without potential Thr glycosylation sites was expressed in the yeast system.     

Structure of type I antifreeze protein and mutants in supercooled water

Many organisms are able to survive subzero temperatures at which bodily fluids would normally be expected to freeze. These organisms have adapted to these lower temperatures by synthesizing antifreeze proteins (AFPs), capable of binding to ice, which make further growth of ice energetically unfavorable. To date, the structures of five AFPs have been determined, and they show considerable sequence and structural diversity. The type I AFP reveals a single 37-residue alpha-helical structure. We have studied the behavior of wild-type type I AFP and two "inactive" mutants (Ala17Leu and Thr13Ser/Thr24Ser) in normal and supercooled solutions of H(2)O and deuterium oxide (D(2)O) to see if the structure at temperatures below the equilibrium freezing point is different from the structure observed at above freezing temperatures. Analysis of 1D (1)H- and (13)C-NMR spectra illustrate that all three proteins remain folded as the temperature is lowered and even seem to become more alpha-helical as evidenced by (13)C(alpha)-NMR chemical shift changes. Furthermore, (13)C-T(2) NMR relaxation measurements demonstrate that the rotational correlation times of all three proteins behave in a predictable manner under all temperatures and conditions studied. These data have important implications for the structure of the AFP bound to ice as well as the mechanisms for ice-binding and protein oligomerization.