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 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.     

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.     

Local ice melting by an antifreeze protein

Antifreeze proteins, AFP, impede freezing of bodily fluids and damaging of cellular tissues by low temperatures. Adsorption-inhibition mechanisms have been developed to explain their functioning. Using in silico Molecular Dynamics, we show that type I AFP can also induce melting of the local ice surface. Simulations of antifreeze-positive and antifreeze-negative mutants show a clear correlation between melting induction and antifreeze activity. The presence of local melting adds a function to type I AFPs that is unique to these proteins. It may also explain some apparently conflicting experimental results where binding to ice appears both quasipermanent and reversible.     

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3₁₀-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.     

Understanding the mechanism of ice binding by type III antifreeze proteins

Type III antifreeze proteins (AFPs) are present in the body fluids of some polar fishes where they inhibit ice growth at subzero temperatures. Previous studies of the structure of type III AFP by NMR and X-ray identified a remarkably flat surface on the protein containing amino acids that were demonstrated to be important for interaction with ice by mutational studies. It was proposed that this protein surface binds onto the (1 0 [\bar 1] 0) plane of ice with the key amino acids interacting directly with the water molecules in the ice crystal. Here, we show that the mechanism of type III AFP interaction with ice crystals is more complex than that proposed previously. We report a high-resolution X-ray structure of type III AFP refined at 1.15 A resolution with individual anisotropic temperature factors. We report the results of ice-etching experiments that show a broad surface coverage, suggesting that type III AFP binds to a set of planes that are parallel with or inclined at a small angle to the crystallographic c-axis of the ice crystal. Our modelling studies, performed with the refined structure, confirm that type III AFP can make energetically favourable interactions with several ice surfaces.     

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.     

Properties,Potentials, and Prospects of Antifreeze Proteins

Antifreeze proteins (AFPs) are a group of proteins that protect organisms from deep freezing temperatures and are expressed in vertebrates, invertebrates, plants, bacteria, and fungi. The nuclear magnetic resonance, x-ray structure, and many spectroscopic studies with AFPs have been instrumental in determining the structure–function relationship. Mutational studies have indicated the importance of hydrophobic residues in ice binding. Various studies have pointed out that the mechanism of AFP action is through its adsorption on the ice surface, which leads to a curved surface, preventing further growth of ice by the “Kelvin effect.” The AFPs have potential industrial, medical, and agricultural application in different fields, such as food technology, preservation of cell lines, organs, cryosurgery, and cold hardy transgenic plants and animals. However, the applications of AFPs are marred by high cost due to low yield. This review deals with the source and properties of AFPs from an angle of their application and their potential. The possibility of production using different molecular biological techniques, which will help increase the yield, is also dealt with.     

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.     

Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270 μmol.     

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.     

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, T_m, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.     

Effect of a mutation on the structure and dynamics of an alpha-helical antifreeze protein in water and ice

One strategy of psychrophilic organisms to survive subzero temperature is to produce antifreeze protein (AFPs), which inhibit the growth of macromolecular ice. To better understand the binding mechanism, the structure and dynamics of several AFPs have been studied by nuclear magnetic resonance (NMR) and X-ray crystallography. The results have shown that different organisms can use diverse structures (alpha-helix, beta-helix, or different globular folds) to achieve the same function. A number of studies have focused on understanding the relationship between the alpha-helical structure of fish type I AFP and its function as an inhibitor of ice growth. The results have not explained whether the 90% activity loss caused by the conservative mutation of two threonines to serines (Thr13Ser/Thr24Ser) is attributable to a change in protein structure in solution or in ice. We examine here the structure and dynamics of the winter flounder type I AFP and the mutant Thr13Ser/Thr24Ser in both solution and solid states using a wide range of NMR approaches. Both proteins remain fully alpha-helical at all temperatures and in ice, demonstrating that the activity change must therefore not be attributable to changes in the protein fold or dynamics but differences in surface properties.     

Antifreeze proteins bind independently to ice.

It has been suggested that cooperative interactions between antifreeze proteins (AFPs) on the ice surfaces are required for complete inhibition of ice crystal growth. To test this hypothesis, a 7-kDa type III AFP was linked through its N-terminus to thioredoxin (12 kDa) or maltose-binding protein (42 kDa). The resultant 20-kDa and 50-kDa fusion proteins were larger in diameter than free AFP and thus precluded any extensive AFP-AFP contacts on the ice surface. Both fusion proteins were at least as active as free AFP at virtually all concentrations tested. By these criteria, AFPs function independently of each other and do not require specific intermolecular interactions to bind tightly to ice.     

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.     

Fluorescence microscopy evidence for quasi-permanent attachment of antifreeze proteins to ice surfaces

Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins.     

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     

Properties, potentials, and prospects of antifreeze proteins

Antifreeze proteins (AFPs) are a group of proteins that protect organisms from deep freezing temperatures and are expressed in vertebrates, invertebrates, plants, bacteria, and fungi. The nuclear magnetic resonance, x-ray structure, and many spectroscopic studies with AFPs have been instrumental in determining the structure-function relationship. Mutational studies have indicated the importance of hydrophobic residues in ice binding. Various studies have pointed out that the mechanism of AFP action is through its adsorption on the ice surface, which leads to a curved surface, preventing further growth of ice by the "Kelvin effect." The AFPs have potential industrial, medical, and agricultural application in different fields, such as food technology, preservation of cell lines, organs, cryosurgery, and cold hardy transgenic plants and animals. However, the applications of AFPs are marred by high cost due to low yield. This review deals with the source and properties of AFPs from an angle of their application and their potential. The possibility of production using different molecular biological techniques, which will help increase the yield, is also dealt with.     

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.