Antifreeze Activity Research Articles

Potential for resistance to freezing by non-virulent bacteria isolated from Antarctica.

Industrial sectors are searching for new compounds to improve the preservation of food and blood, human tissues, and fuels used at low temperatures. Antarctic microorganisms have mechanisms to overcome injuries caused by low temperatures, making them sources of compounds with antifreeze activity. However, it is mandatory that such compounds do not pose a risk to human health. The present study evaluated the potential of Antarctic bacteria to resist freezing, produce virulence factors, their tolerance to physiological pHs/temperature and resistance to antibiotics. Sixty-five isolates were tested for antifreeze compound production, among which, 31 grew after the test. Of these, 3 strains of Arthrobacter sp. (356, 358 and 443), one Psychromonas arctica (ESH238) and one unidentified strain (363) showed positive results for hemolytic activity. Psychrobacter sp. 456 showed proteinase activity. None of the isolates showed resistance to the antibiotics. All isolates were able to grow in one of the three pHs (4, 7 and 8) and/or temperature (36, 38 and 40 ºC). Antarctic bacterial present potential for the production of antifreeze compounds and may be considered safe in industrial processes. The characterization of the genes responsible for virulence factors should be carried out to reinforce the potential applicability of such bacteria.

Transcriptomics of a Greenlandic Snailfish Reveals Exceptionally High Expression of Antifreeze Protein Transcripts.

Polar fishes have evolved antifreeze proteins (AFPs) that allow them to survive in subzero temperatures. We performed deep transcriptomic sequencing on a postlarval/juvenile variegated snailfish, Liparis gibbus (Actinopterygii: Scorpaeniformes: Cottoidei: Liparidae), living in an iceberg habitat (−2°C) in Eastern Greenland and report detection of highly expressed transcripts that code for putative AFPs from 2 gene families, Type I and LS-12-like proteins (putative Type IV AFPs). The transcripts encoding both proteins have expression levels among the top <1% of expressed genes in the fish. The Type I AFP sequence is different from a reported Type I AFP from the same species, possibly expressed from a different genetic locus. While prior findings from related adult sculpins suggest that LS-12-like/Type IV AFPs may not have a role in antifreeze protection, our finding of very high relative gene expression of the LS-12-like gene suggests that highly active transcription of the gene is important to the fish in the iceberg habitat and raises the possibility that weak or combinatorial antifreeze activity could be beneficial. These findings highlight the physiological importance of antifreeze proteins to the survival of fishes living in polar habitats.

Identification and Functional Characterization of Antifreeze Protein and Its Mutants in Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae) Larvae Under Cold Stress.

Dendroctonus armandi (Tsai and Li) (Coleoptera: Curculionidae: Scolytinae) is considered to be the most destructive forest pest in the Qinling and Bashan Mountains of China. Low winter temperatures limit insect's populations, distribution, activity, and development. Insects have developed different strategies such as freeze-tolerance and freeze-avoidance to survive in low temperature conditions. In the present study, we used gene cloning, real-time polymerase chain reaction (PCR), RNA interference (RNAi), and heterologous expression to study the function of the D. armandi antifreeze protein gene (DaAFP). We cloned the 800 bp full-length cDNA encoding 228 amino acids of DaAFP and analyzed its structure using bioinformatics analysis. The DaAFP amino acid sequence exhibited 24-86% similarity with other insect species. The expression of DaAFP was high in January and in the larvae, head, and midgut of D. armandi. In addition, the expression of DaAFP increased with decreasing temperature and increasing exposure time. RNAi analysis also demonstrated that AFP plays an important role in the cold tolerance of overwintering larvae. The thermal hysteresis and antifreeze activity assay of DaAFP and its mutants indicated that the more regular the DaAFP threonine-cystine-threonine (TXT) motif, the stronger the antifreeze activity. These results suggest that DaAFP plays an essential role as a biological cryoprotectant in overwintering D. armandi larvae and provides a theoretical basis for new pest control methods.

Isolation of novel wheat bran antifreeze polysaccharides and the cryoprotective effect on frozen dough quality

Different wheat bran antifreeze polysaccharides (WBAP) were first isolated by ice shell separation method. The composition and antifreeze activity of varied WBAP were characterized and their cryoprotective effects on frozen dough were comprehensively studied with the emphasis on ice crystals, yeast and gluten proteins. WBAP with enriched arabinoxylan (AX) of high arabinofuranose/xylopyranose ratio (A/X) showed superior effect on inhibiting ice recrystallization compared with WBAP with low A/X. WBAP could significantly improve the quality of frozen dough steamed bread, and the effect coincided with antifreeze activity. WBAP effectively inhibited the ice recrystallization and altered the shape of ice crystal from cubic to lamellate in frozen dough, consequently better preserving the yeast viability and dough structure. WBAP alleviated the depolymerization of glutenin macropolymers, and was conductive in developing more homogeneous and less distorted gluten network structures in frozen dough. The current study can provide a theoretical basis for the exploitation of WBAP as a technofunctional improver for frozen dough.

Comparative N-glycoproteomic analysis of Tibetan and lowland chicken fertilized eggs: Implications on proteins biofunction and species evolution.

The characterization and functionality of protein glycosylation among different related species are of common interest. Herein, non-standard quantification and N-glycosylation enrichment technology combined with ultra-high liquid chromatography-tandem mass spectrometry were used to establish detailed N-glycoproteomics of fertilized eggs, and quantitatively compared between Tibetan and lowland chicken. A total of 396N-glycosites from 143 glycoproteins were found. Specifically, compared with lowland chicken egg white, 32N-glycosites of 22 glycoproteins were up-regulated and 57N-glycosites of 25 glycoproteins were down-regulated in Tibetan chicken egg white. Also, 137N-glycosites in 72 glycoproteins showed much higher-degree glycosylation and 36N-glycosites in 15 glycoproteins displayed lower-degree glycosylation in Tibetan chicken egg yolk than those in lowland chicken egg yolk. Through bioinformatic analysis, these varied glycoproteins werehighlyassociated with antifreeze activity, hypoxia adaptation, coagulation cascade, and binding/immunity activities, which may be related to plateau hypoxia and cold stress. PRACTICAL APPLICATIONS: These findings provide a new insight on the role of biological egg N-glycoproteins related to environmental adaptation and evolution, which may be further applied in improving egg processing and human health, by developing biomolecules for food and medical industry.

High Water Density at Non-Ice-Binding Surfaces Contributes to the Hyperactivity of Antifreeze Proteins.

Antifreeze proteins (AFPs) can bind to ice nuclei thereby inhibiting their growth and their hydration shell is believed to play a fundamental role. Here, we use molecular dynamics simulations to characterize the hydration shell of four moderately-active and four hyperactive AFPs. The local water density around the ice-binding-surface (IBS) is found to be lower than that around the non-ice-binding surface (NIBS) and this difference correlates with the higher hydrophobicity of the former. While the water-density increase (with respect to bulk) around the IBS is similar between moderately-active and hyperactive AFPs, it differs around the NIBS, being higher for the hyperactive AFPs. We hypothesize that while the lower water density at the IBS can pave the way to protein binding to ice nuclei, irrespective of the antifreeze activity, the higher density at the NIBS of the hyperactive AFPs contribute to their enhanced ability in inhibiting ice growth around the bound AFPs.

Differential Hydration of Ice‐Binding Surface of Globular and Hyperactive Antifreeze Proteins

AbstractLike hyperactive antifreeze proteins (AFPs), globular AFPs also illustrate thermal hysteresis by specific binding to ice. The ice‐binding surface (IBS) topology and residue preferences are different between globular AFPs and hyperactive AFPs. This study explores the hydration water ordering and its temperature and length‐scale dependencies for type‐III AFPs, and the results with hyperactive AFP and non‐AFPs are compared. The hydration layer of the IBS of globular AFP is semiclathrate‐like, which is different from the hyperactive AFPs, as it is reported to be ice‐like. In a similar environment, the solvation water surrounding the non‐ice‐binding surface (non‐IBS) is mostly liquid‐like. Surprisingly, the non‐IBS is appreciably dissimilar from the non‐AFP in terms of hydration pattern. The amino acid compositions of the non‐IBS are in contrast with the IBS. Protein–water hydrogen bonding patterns between the IBS and non‐IBS of AFP are also different. These consequences suggest the collective function of non‐IBS and IBS in the antifreeze activity. The different nature of the first solvation shell water ordering around the IBS of type‐III AFP and hyperactive insect AFP implicates a differential design of IBS that may account for their observed difference in antifreeze activity.

Cold denaturation induced helix-to-helix transition and its implication to activity of helical antifreeze protein

Study of cold denaturation is relevant for understanding the freeze tolerant activity of organism at sub-zero environment. The proteins responsible for the survival of these organisms contain abundance of alpha-helix. Here we study the effect of cold temperature on the alpha-helical region of a widely used model system, Trp-cage miniprotein by means of extensive atomistic molecular dynamics simulations in conjunction with replica exchange scheme. We find a helix-to-helix transition when the usual ~3.59 alpha-helix transforms to ~3.7 helix upon cooling below ~230 K. This transition is primarily driven by enthalpy gain from water-water interactions with the significant enhancement of low-density liquid (LDL)-like water at cold temperature. Because the transition from ~3.59-helix to ~3.7-helix occurs at cold temperature, it may be accompanied by cold denaturation of protein. Like the cold denatured state of Trp-cage, type-1 antifreeze proteins (AFP) also have ~3.7 or more residues per helical turn. However, unlike Trp-cage, AFP has the 3.7-helix in its biological active form. Such helical arrangement in AFP facilitates the hydrogen bonding with the surrounding clathrate or ice-like water molecules, which is crucial for the antifreeze activity of AFP.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP’s unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.

Thermal hysteresis activity of antifreeze proteins: A model based on fractional statistics theory of adsorption

Antifreeze proteins (AFPs) adsorb to the surface of embryonic ice crystals to prevent their growth. The protein-ice adsorption lowers the freezing point of the solution. Then, a thermal hysteresis can be defined as the difference between the melting and freezing temperatures. This quantity is a measure of the antifreeze protein activity. In this sense, there exists evidence that the antifreeze activity enhances with increasing the area/length of the ice-binding sites. In order to interpret this thermal hysteresis behavior, we introduce a two-dimensional adsorption model based on fractional statistics theory. The analytical expressions are obtained in terms of an exclusion parameter, which depend on the structure of the protein and area of the ice-binding sites. By using the model, thermal hysteresis activity is calculated for AFPs of different size, shape and number of active sites. The theoretical results show a good qualitative agreement with reported experimental data in the literature.

Characterization of microbial antifreeze protein with intermediate activity suggests that a bound-water network is essential for hyperactivity

Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein’s ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

Brassica juncea leaf cuticle proteome analysis shows myrosinase protein, antifreeze activity, and post-translationally modified secretory proteins

Plant cuticle, the site of perception of stress signals, is an extracellular hydrophobic barrier that covers the epidermis of the above-ground parts. This lipidic layer has been explored for its cutin and wax composition. However, reports on the cuticle proteins are scanty. Therefore, leaf cuticle proteins of Brassica juncea isolated using organic solvents (chloroform-methanol, 2:1(v/v)) were analyzed using gel based and quantitative shotgun proteomics. Out of 615 proteins identified, 27% (169) had signal peptides supporting extracellular localization. Bioinformatics tool, QuickGO predicted the involvement of these proteins in catabolism (21%), peptidase activity (13%), oxidoreductase (12%), defense response (9%), fatty acid binding (9%), nutrient reservoir activity (8%), chitin binding (7%) and lipid transport (2%). Myrosinase-catalyzed glucosinolate hydrolysis releases bioactive compounds, which contribute to plant defense. This system is termed as “mustard oil bomb”. Myrosinase and its associating protein, GDSL esterase/lipase ESM1 (involved in cuticle structuring and defense) were detected in the cuticle. GDSL-esterase/lipase ESM1 and β-glucanase (an antifreeze protein) showed in vitro activity. Analysis of cuticle extract by nanoliter osmometer-phase contrast microscopy detected antifreeze activity due to non-protein component. Post-translational modification analysis using PTM viewer predicted N-glycosylation (66%), N-terminal proteolysis (40%), and phosphorylation (32%) to be the dominant modification in the classical secretory proteins. N-glycosylation of myrosinase and GDSL esterase/lipase, ESM1 was confirmed by Con A affinoblotting. This study not only identified leaf cuticle proteins, but also laid the foundation for exploring the extracellular glucosinolate-myrosinase system, PTM crosstalk, and antifreeze activity as stress adaptive strategies in B. juncea.

Crystal waters on the nine polyproline type II helical bundle springtail antifreeze protein from Granisotoma rainieri match the ice lattice.

A springtail (Collembola) identified as Granisotomarainieri was collected from snow in Hokkaido, Japan, in late winter when nighttime temperatures were below zero. Extracts of these arthropods showed antifreeze activity by shaping ice crystals and stopping their growth. The glycine-rich proteins responsible for this freezing point depression were isolated by ice-affinity purification and had principal masses of ~6.9 and 9.6kDa. We identified a transcript for a 9.6-kDa component and produced it as a His-tagged recombinant protein for structural analysis. Its crystal structure was solved to a resolution of 1.21Å and revealed a polyproline type II helical bundle, similar to the six-helix Hypogastruraharveyi AFP, but with nine helices organized into two layers held together by an extensive network of hydrogen bonds. One of the layers is flat, regular, and hydrophobic and likely serves as the ice-binding side. Although this surface makes close protein-protein contacts with its symmetry mate in the crystal, it has bound chains of waters present that resemble those on the basal and primary prism planes of ice. Molecular dynamic simulations indicate most of these crystal waters would preferentially occupy these sites if exposed to bulk solvent in the absence of the symmetry mate. These prepositioned waters lend further support to the ice-binding mechanism in which AFPs organize ice-like waters on one surface to adsorb to ice. DATABASES: Structural data are available in the Protein Data Bank under the accession number 7JJV. Transcript data are available in GenBank under accession numbers MT780727, MT780728, MT780729, MT780730, MT780731 and MT985982.

Ice recrystallization inhibition activity varies with ice-binding protein type and does not correlate with thermal hysteresis

Ice-binding proteins (IBPs) inhibit the growth of ice through surface adsorption. In some freeze-resistant fishes and insects, circulating IBPs serve as antifreeze proteins to stop ice growth by lowering the freezing point. Plants are less able to avoid freezing and some use IBPs to minimize the damage caused in the frozen state by ice recrystallization, which is the growth of large ice grains at the expense of small ones. Here we have accurately and reproducibly measured the ice recrystallization inhibition (IRI) activity of over a dozen naturally occurring IBPs from fishes, insects, plants, and microorganisms using a modified ‘splat’ method on serial dilutions of IBPs whose concentrations were determined by amino acid analysis. The endpoint of IRI, which was scored as the lowest protein concentration at which no recrystallization was observed, varied for the different IBPs over two orders of magnitude from 1000 nM to 5 nM. Moreover, there was no apparent correlation between their IRI levels and reported antifreeze activities. IBPs from insects and fishes had similar IRI activity, even though the insect IBPs are typically 10x more active in freezing point depression. Plant IBPs had weak antifreeze activity but were more effective at IRI. Bacterial IBPs involved in ice adhesion showed both strong freezing point depression and IRI. Two trends did emerge, including that basal plane binding IBPs correlated with stronger IRI activity and larger IBPs had higher IRI activity.

Impact of borate on structure of antifreeze glycoproteins

Antifreeze glycoproteins (AFGPs) facilitate the survival of various organisms in the polar region by preventing internal ice accumulation via an adsorption-inhibition mechanism. Inhibition of AFGP antifreeze activity by the borate buffers has been widely acknowledged as the direct experimental evidence supporting the hydroxyl, rather than methyl, binding mechanism. On the other hand, perturbation of borate binding on the AFGP configuration, which might have considerable influence on the binding efficiency of not only the hydroxyl but also the methyl groups, has rarely been quantitatively examined. Herein we studied, using molecular dynamics simulations, the perturbation on the configuration of a solvated AFGP8 protein induced by the binding of one single borate anion. Near the freezing point, this binding not only makes the disaccharide groups adjacent to the borate-binding disaccharide close to each other but also affects the entire AFGP8 conformation. The structural changes induced by the binding of borate on different disaccharide sidechains exhibit clear site-specificities and the effect of borate binding on the structural changes is significantly reduced at higher temperatures. Our study is valuable for further understanding the relationship between the structure and antifreeze activity of these antifreeze glycoproteins.

Functional characterization of VviDHN2 and VviDHN4 dehydrin isoforms from Vitis vinifera (L.): An in silico and in vitro approach

Dehydrins, a family of hydrophilic and intrinsically disordered proteins, are a subgroup of late embryogenesis abundant proteins that perform different protective roles in plants. Although the transition from a disordered to an ordered state has been associated with dehydrin function or interactions with specific partner molecules, the question of how the primary and secondary dehydrin protein structure is related to specific functions or target molecule preferences remains unresolved. This work addresses the in silico sequencing analysis and in vitro functional characterization of two dehydrin isoforms, VviDHN2 and VviDHN4, from Vitis vinifera. Conformational changes suggest potential interactions with a broad range of molecules and could point to more than one function. The in silico analysis showed differences in conserved segments, specific amino acid binding sequences, heterogeneity of structural properties and predicted sites accessible for various post-translational modifications between the sequence of both dehydrins. Moreover, in vitro functional analysis revealed that although they both showed slight antifungal activity, only VviDHN4 acts as a molecular shield that protects proteins from freezing and dehydration. VviDHN4 also demonstrated high potential as a chaperone and reactive oxygen species scavenger, in addition to presenting antifreeze activity, all of which confirms its multifunctional nature. Our findings highlight the significant role of Y-segments and the differential and specific amino acid composition of less conserved segments that are rich in polar/charged residues between S- and K-segments, coupled with post-translational modifications, in modulating and switching dehydrin biological function.

Novel antifreeze activities of pegylated type III antifreeze proteins

Novel antifreeze activities of pegylated type III antifreeze proteins

Genetic Algorithm Approach for the Optimization of Protein Antifreeze Activity Using Molecular Simulations

Antifreeze proteins (AFPs) are of much interest for their ability to inhibit ice growth at low concentrations. In this work, we present a genetic algorithm for the in silico design of AFP mutants with improved antifreeze activity, measured as the predicted thermal hysteresis at a fixed concentration, ΔTC. Central to the algorithm is our recently developed neural network method for predicting ΔTC from molecular simulations [Kozuch et al., PNAS, 115, 13252 (2018)]. Applying the algorithm to three structurally diverse AFPs, wfAFP, rQAE, and RiAFP, we find that significantly improved mutants are discovered for rQAE and RiAFP. Testing of the optimized mutants shows an increase in ΔTC of 0.572 ± 0.11 K (262 ± 50.6%) and 1.33 ± 0.14 K (39.9 ± 4.19%) over the native structures for rQAE and RiAFP, respectively. Structural analysis of the optimized mutants reveals that the algorithm is able to exploit two pathways for enhancing the predicted antifreeze activity of the mutants: (1) increasing the local order of surface waters by encouraging the formation of internal water channels in the protein and (2) increasing the total ice-binding area by improving the planar structure of the ice-binding surface. Additionally, analysis of all mutants explored by the algorithm reveals that a subset of residues, mainly nonpolar, are particularly helpful in improving antifreeze activity at the ice-binding surface.

Production, structure-function relationships, mechanisms, and applications of antifreeze peptides.

Growth of ice crystals can cause serious problems, such as frozen products deterioration, road damage, energy losses, and safety risks of human beings. Antifreeze peptides (AFPs), a healthy and effective cryoprotectant, have great potential as ice crystal growth inhibitors for a variety of frozen products. In this review, methods and technologies for the production, purification, evaluation, and characterization of AFPs are comprehensively summarized. First, this review describes the preparation of AFPs, including the methods of enzymatic hydrolysis, chemical synthesis, and microbial fermentation. Next, this review introduces the major methods by which to evaluate AFPs' antifreeze activity, including nanoliter osmometer, differential scanning calorimetry, splat-cooling, the biovaluation model, and novel technology. Moreover, this review presents an overview of the molecular characteristics, structure-function relationships, and action mechanisms of AFPs. Furthermore, advances in the application of AFPs to frozen food, including frozen dough, meat products, fruits, vegetable products, and dairy, are summarized andholistically analyzed. Finally, challenges of AFPs and future perspectives on their use are also discussed. An understanding of the production, structure-function relationships, mechanisms and applications of AFPs provides inspiration for further research into the use of AFPs in food science and food nutrition applications.

Characterization and identification of a fraction from silver carp (Hypophthalmichthys molitrix) muscle hydrolysates with cryoprotective effects on yeast

This study was aimed to characterize and identify a fraction from silver carp muscle hydrolysates (SCMHs) with cryoprotective effects on yeast. The SCMHs were obtained from the hydrolysis of silver carp (Hypophthalmichthys molitrix) muscle with Protamex at 50 °C for 15–240 min. The addition of SCMH generated from 30 min of proteolysis (SCMH-30) led to an increase in the yeast survival rate from 0 – 1% (control) to 82–91% after 1–2 freeze-thaw cycles. The SCMH-30 was further separated into two fractions using gel filtration chromatography. The fraction F2 with lower average molecular weight exhibited better cryoprotective effect on yeast, which was further characterized via HPLC-MS/MS. Among a total of 2500 identified peptides, 90% of them had molecular weight between 1000 and 3000 Da, which were mainly derived from parvalbumins. The selected primary peptides were rich in glutamic and aspartic acid residues and exhibited structural and sequential similarities with type IV fish antifreeze proteins, which likely contributed to the high antifreeze activity of F2 from SCMH-30. Results from this study provided an approach for development of effective cryoprotectants using SCMHs and helped to understand the underlying mechanism governing cryoprotective effect of SCMH-30 on yeast.