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.     

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.     

Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges

A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is two-state, the psychrophilic type III AFP has a much lower thermodynamic stability at 25 degrees C, approximately 3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0 degrees C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (approximately 1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25 degrees C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0 degrees C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.     

Effect of annealing time of an ice crystal on the activity of type III antifreeze protein

Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8). TH of nfeAFP8 dramatically decreased when using a fast cooling rate (0.20 degrees C x min(-1)). It also decreased with increasing seed crystal size under a slow cooling rate (0.01 degrees C x min(-1)), but such dependence was not detected under the fast cooling rate. TH was enhanced 1.4- and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25 degrees C below T(m), respectively. After annealing for 2 h at 0.25 degrees C below T(m), TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2-3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption-inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2-3 h time scale, which results in the TH dependence on the annealing time.     

Spruce budworm antifreeze protein: changes in structure and dynamics at low temperature

Antifreeze proteins (AFPs) prevent the growth of ice, and are used by some organisms that live in sub-zero environments for protection against freezing. All AFPs are thought to function by an adsorption inhibition process. In order to elucidate the ice-binding mechanism, the structures of several AFPs have been determined, and have been shown to consist of different folds. Recently, the first structures of the highly active insect AFPs have been characterized. These proteins have a beta-helix structure, which adds yet another fold to the AFP family. The 90-residue spruce budworm (Choristoneura fumiferana) AFP consists of a beta-helix with 15 residues per coil. The structure contains two ranks of aligned threonine residues (known as the TXT motif), which were shown by mutagenesis experiments to be located in the ice-binding face. In our previous NMR study of this AFP at 30 degrees C, we found that the TXT face was not optimally defined because of the broadening of NMR resonances potentially due to weak oligomerization. We present here a structure of spruce budworm AFP determined at 5 degrees C, where this broadening is reduced. In addition, the 1H-15N NMR dynamics of the protein were examined at 30 degrees C and 5 degrees C. The results show that the spruce budworm AFP is more structured at 5 degrees C, and support the general observation that AFPs become more rigid as the temperature is lowered.     

The stability during low-temperature storage of an antifreeze protein isolated from the roots of cold-acclimated carrots

Natural antifreeze proteins (AFPs) not only inhibit freezing at high subzero temperatures; they have the additional properties of inhibiting the recrystallization of ice during warming and of preventing devitrification. The natural AFP that occurs in the roots of cold-acclimated carrots can be extracted reasonably simply and is non-toxic: it was selected for study as a possible ingredient of the vitrification mixtures that are being developed for use in tissue cryopreservation. For this application, it would be essential for the AFP to remain active during prolonged storage at very low temperatures. For logistic reasons, it would also be essential to have an effective method of storage of the purified AFP itself. In this study, carrot AFP was isolated and purified, and its ability to inhibit recrystallization was monitored over 40 weeks of storage at -80 or -196 degrees C. The data revealed a progressive decrease in activity during storage, reaching half the original activity in 10-20 weeks and only 2-3% of the original activity at 40 week. These data suggest that carrot AFP will not be effective in tissue cryopreservation.     

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.     

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.     

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.     

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.     

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.     

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.     

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.     

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.     

Solution structure of a recombinant type I sculpin antifreeze protein

We have determined the solution structure of rSS3, a recombinant form of the type I shorthorn sculpin antifreeze protein (AFP), at 278 and 268 K. This AFP contains an unusual sequence of N-terminal residues, together with two of the 11-residue repeats that are characteristic of the type I winter flounder AFP. The solution conformation of the N-terminal region of the sculpin AFP has been assumed to be the critical factor that results in recognition of different ice planes by the sculpin and flounder AFPs. At 278 K, the two repeats units (residues 11-20 and 21-32) in rSS3 form a continuous alpha-helix, with the residues 30-33 in the second repeat somewhat less well defined. Within the N-terminal region, residues 2-6 are well defined and helical and linked to the main helix by a more flexible region comprising residues A7-T11. At 268 K the AFP is overall more helical but retains the apparent hinge region. The helical conformation of the two repeats units is almost identical to the corresponding repeats in the type I winter flounder AFP. We also show that while tetracetylated rSS3 has antifreeze activity comparable to the natural AFP, its overall structure is the same as that of the unacetylated peptide. These data provide some insight into the structural determinants of antifreeze activity and should assist in the development of models that explain the recognition of different ice interfaces by the sculpin and flounder type I AFPs.     

Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant

Plants' distribution and productivity are adversely affected by low temperature (LT) stress. LT induced proteins were analyzed by 2-DE-nano-LC-MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub. Seedlings were subjected to direct freezing stress (-5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. LT induced secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide an insight in probable LT induced signaling in the secretome.     

Systematic size study of an insect antifreeze protein and its interaction with ice

Because of their remarkable ability to depress the freezing point of aqueous solutions, antifreeze proteins (AFPs) play a critical role in helping many organisms survive subzero temperatures. The beta-helical insect AFP structures solved to date, consisting of multiple repeating circular loops or coils, are perhaps the most regular protein structures discovered thus far. Taking an exceptional advantage of the unusually high structural regularity of insect AFPs, we have employed both semiempirical and quantum mechanics computational approaches to systematically investigate the relationship between the number of AFP coils and the AFP-ice interaction energy, an indicator of antifreeze activity. We generated a series of AFP models with varying numbers of 12-residue coils (sequence TCTxSxxCxxAx) and calculated their interaction energies with ice. Using several independent computational methods, we found that the AFP-ice interaction energy increased as the number of coils increased, until an upper bound was reached. The increase of interaction energy was significant for each of the first five coils, and there was a clear synergism that gradually diminished and even decreased with further increase of the number of coils. Our results are in excellent agreement with the recently reported experimental observations.     

Lateral transfer of a lectin-like antifreeze protein gene in fishes

Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs) that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven) cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.