High Antifreeze Activity Research Articles

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.

Hydrophobic ice-binding sites confer hyperactivity of an antifreeze protein from a snow mold fungus.

Snow mold fungus, Typhula ishikariensis, secretes seven antifreeze protein isoforms (denoted TisAFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, TisAFP8, at 1.0 Å resolution is presented. TisAFP8 folds into a right-handed β-helix accompanied with a long α-helix insertion. TisAFP8 exhibited significantly high antifreeze activity that is comparable with other hyperactive AFPs, despite its close structural and sequence similarity with the moderately active isoform TisAFP6. A series of mutations introduced into the putative ice-binding sites (IBSs) in the β-sheet and adjacent loop region reduced antifreeze activity. A double-mutant A20T/A212S, which comprises a hydrophobic patch between the β-sheet and loop region, caused the greatest depression of antifreeze activity of 75%, when compared with that of the wild-type protein. This shows that the loop region is involved in ice binding and hydrophobic residues play crucial functional roles. Additionally, bound waters around the β-sheet and loop region IBSs were organized into an ice-like network and can be divided into two groups that appear to mediate separately TisAFP and ice. The docking model of TisAFP8 with the basal plane via its loop region IBS reveals a better shape complementarity than that of TisAFP6. In conclusion, we present new insights into the ice-binding mechanism of TisAFP8 by showing that a higher hydrophobicity and better shape complementarity of its IBSs, especially the loop region, may render TisAFP8 hyperactive to ice binding.

Inhibition of Gas Hydrate Nucleation and Growth: Efficacy of an Antifreeze Protein from the Longhorn Beetle Rhagium mordax

Antifreeze proteins (AFPs) are characterized by their ability to protect organisms from subfreezing temperatures by preventing tiny ice crystals in solution from growing as the solution is cooled below its freezing temperature. This inhibition of ice growth is called antifreeze activity, and in particular, certain insect AFPs show very high antifreeze activity. Recent studies have shown AFPs to be promising candidates as green and environmentally benign inhibitors for gas hydrate formation. Here we show that an insect antifreeze protein from the longhorn beetle, Rhagium mordax (RmAFP1), the most potent protein yet found for freezing inhibition, can inhibit methane hydrates as effectively as the synthetic polymeric inhibitor polyvinylpyrrolidone (PVP). In high pressure rocking cell experiments, onset hydrate nucleation temperatures and growth profiles showed repeatable results. RmAFP1 clearly showed inhibition of hydrates compared to amino acids (l-valine and l-threonine) and the protein bovine serum album...

Long-range protein-water dynamics in hyperactive insect antifreeze proteins.

Antifreeze proteins (AFPs) are specific proteins that are able to lower the freezing point of aqueous solutions relative to the melting point. Hyperactive AFPs, identified in insects, have an especially high ability to depress the freezing point by far exceeding the abilities of other AFPs. In previous studies, we postulated that the activity of AFPs can be attributed to two distinct molecular mechanisms: (i) short-range direct interaction of the protein surface with the growing ice face and (ii) long-range interaction by protein-induced water dynamics extending up to 20 Å from the protein surface. In the present paper, we combine terahertz spectroscopy and molecular simulations to prove that long-range protein-water interactions make essential contributions to the high antifreeze activity of insect AFPs from the beetle Dendroides canadensis. We also support our hypothesis by studying the effect of the addition of the osmolyte sodium citrate.

Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12

The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1°C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30% similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6h of growth at -12°C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refoldin vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08°C.

Effects antifreeze peptides on the thermotropic properties of a model membrane

In this paper, we report on the effect of short segments of type I antifreeze protein (AFP I) on the thermotropic properties of a model membrane. Two different types of dimyristoylphosphatidylcholine model membranes were used, multilamellar vesicles and small unilamellar vesicles. The membrane properties were studied by differential scanning calorimetry (DSC) and fluorescence anisotropy. With the incorporation of AFP I and its short segments, the order of the model membrane increased both in the gel state and in the liquid crystalline state. The interaction of AFPs with the model membrane caused a shift in the phase transition to lower temperatures, which is accompanied by a broadening of the DSC thermogram. This preferential stabilization to a more ordered phase by the AFPs could be due to ordering the hydrophobic membrane core and separation into domains. Overall, this approach of employing short segments of AFP I simplifies the correlation between antifreeze protein characteristics and the effect of these parameters on the interaction mechanism of AFP with cell membranes. The success of this approach can lead to the identification of short peptides with high antifreeze activity.

Activity of short segments of Type I antifreeze protein

In this work, we present a study on the antifreeze activity of short segments of a Type I antifreeze protein, instead of the whole protein. This approach simplifies the correlation between antifreeze protein characteristics, such as hydrophilicity/hydrophobicity, and the effect of these characteristics on the antifreeze mechanism. Three short polypeptides of Type I AFP have been synthesized. Their antifreeze activity and interactions with water and ice crystals have been analyzed by various techniques such as circular dichroism spectroscopy, X-ray diffraction, differential scanning calorimetry, and osmometry. It is shown that one short segment of Type I AFP has an antifreeze activity of about 60% of the native protein activity. In this work, we demonstrate that short segments of Type I AFPs possess nonzero thermal hysteresis and result in modifications in the growth habits and growth rates of ice. This approach enables the preparation of large quantities of short AFP segments at low cost with high antifreeze activity, and opens the possibility of developing the commercial potential of AFPs.

Genetic studies of antifreeze proteins and their correlation with winter survival in wheat

Antifreeze proteins (AFPs) accumulate in the leaves of winter cereals during cold acclimation, where they may inhibit ice recrystallization during freezing and thawing cycles and provide nonspecific disease resistance. In this study, 21 wheat chromosome substitution lines and the parental lines Chinese Spring and Cheyenne wheat were used to determine the heritability of AFPs and the relationship between the accumulation of AFPs and winter survival. In cold-acclimated lines, antifreeze activity in leaf apoplastic extracts ranged from 1 (low) to 5 (high) with an average value of 3.2, and the accumulation of apoplastic proteins ranged from 30 μg (g FW)-1 to 115 μg (g FW)-1 with a mean value of 70 μ (g FW)-1. Examination of the individual lines revealed that Cheyenne chromosomes 5B and 5D carry major regulatory genes that increase both antifreeze activity and the accumulation of antifreeze proteins in plants grown at low temperature. Substitution lines carrying Cheyenne chromosomes 2A, 3A, 6B, and 7A exhibited lower freezing tolerance and also showed a marked decrease in the accumulation of specific AFPs during cold acclimation. Antifreeze activity and apoplastic protein content were not correlated with freezing tolerance (defined as % survival at -11 °C), but they were both significantly and positively correlated with winter field survival rates. Antifreeze activity (positively correlated) and total leaf fresh weight (negatively correlated) together accounted for about 55% of the variation in winter survival, indicating that high antifreeze activity and slow vegetative growth at low temperature are both important quantitative traits for winter survival.

Maintenance of the supercooled state in overwintering pyrochroid beetle larvae, Dendroides canadensis : role of hemolymph ice nucleators and antifreeze proteins

Freeze-avoiding fire-colored beetle larvae, Dendroides canadensis, were monitored seasonally to explore the role of endogenous hemolymph ice nucleators and antifreeze proteins on the maintenance of supercooling. In preparation for overwintering, D. canadensis depressed hemolymph ice nucleator activity and increased thermal hysteresis activity [mean value circa 0. 5 °C (summer) versus circa 5 °C (midwinter)] resulting in decreased larval and hemolymph supercooling points [−7 °C (summer) versus −20 °C (midwinter)]. Results of gel filtration chromatography, flotation ultracentifugation and quantitative investigation of ice nucleator activity using hemolymph from summer and winter collected larvae strongly suggest that highly active protein and lipoprotein ice nucleators are removed in preparation for overwintering. Additions of either purified antifreeze proteins or midwinter hemolymph with high antifreeze protein activity to a mixture of protein or lipoprotein ice nucleators isolated from D. canadensis hemolymph inhibited the activity of these nucleators. This suggests that in addition to seasonal removal, inhibition of hemolymph ice nucleators by antifreeze proteins contributes to seasonal increases in hemolymph supercooling capacity.

Antifreeze Activities of Various Food Components

The antifreeze activities of various food components were measured by differential scanning calorimetry (DSC), and their immobilizing activities for water molecules were measured by nuclear magnetic resonance (NMR). The antifreeze activities of saccharides that consisted of glucose were higher than those of the others. In salts, those which possessed high ionic charge had high antifreeze activities. The DSC results were consistent with the NMR observations, that is, food components which had high antifreeze activity also had high immobilizing activity for water molecules. In water-soluble amino acids, a few amino acids which formed no eutectic mixture above -20 °C had especially high antifreeze activities.