Antifreeze Activity Research Articles

Mechanism of Binding of Polyproline to Ice via Interfacial Water: An Experimental and Theoretical Study.

The molecular mechanism underlying inhibition of ice growth by polyproline (PPro), a minimal antifreeze glycoprotein mimic, remains unclear. In this work, the change in the structure of water during the growth of ice in PPro solutions was investigated using a combination of near-infrared spectroscopy and molecular dynamics (MD) simulations. The results show that only high concentrations of PPro solutions can effectively inhibit ice growth, as indicated by the variation in the spectral intensity of ice with time. When PPro exhibits an antifreeze effect, the spectral intensity of hydrated water associated with PPro in a solution is weakened. The experiments and MD simulations reveal that the quantity of the interfacial water between the ice crystal and the hydrophobic groups of PPro progressively reaches a plateau. Most significantly, we present clear evidence that the stable existence of this interfacial water is critical for the antifreeze activity of PPro.

Development and Implementation of Microbial Antifreeze Protein Based Coating for Anti‐Icing

AbstractIce formation on a solid surface is a major challenge in industrial applications, it causes higher energy consumption and performance deterioration and may lead to catastrophic results. The preparation of anti‐icing surfaces to prohibit ice accumulation on a surface is crucial to reduce operational costs and to extend the surface's lifetime. The utilization of cryoprotectants to obtain anti‐icing surfaces is an effective method and is applicable in multiple fields. Antifreeze proteins (AFPs) are natural cryoprotectants to obtain anti‐icing surfaces, which have the ability to decrease the freezing point and to prevent ice‐crystal growth via thermal hysteresis (TH) and ice recrystallization inhibition (IRI). This study reports the molecular cloning, expression, and production of AFP protein from Escherichia coli (E. Coli). This wok also demonstrates the activity of coated AFP on aluminum surfaces. The expressed AFP is immobilized on aluminum surfaces treated by oxygen plasma. The coated AFP exhibits promising antifreeze activity with a high anti‐icing ability on aluminum surfaces in the size of evaporator fins (40 × 40 cm). The outcome of this study provides new insights into the biotechnological implementation of AFPs to various industrial applications for energy‐saving and higher performance.

The cryoprotective activity of tilapia skin collagen hydrolysate and the structure elucidation of its antifreeze peptide

Antifreeze peptides from fish byproducts have the potential to act as suitable and safe cryoprotectants, replacing synthetic additives currently utilized in the food industry. The main focus of this study was to explore the cryoprotective activity of antifreeze peptides from collagen hydrolysate of tilapia (Oreochromis niloticus) skin and deduce the relationship between its physio-chemical characteristics and antifreeze activity. Our results showed that the antifreeze peptides from tilapia skin collagen hydrolysate (TSCP) by alcalase had a lower molecular weight of <2500 Da, higher content of hydrophilic amino acids, and thermal hysteresis activity was 0.50 °C. TSCP33, the identified novel cryoprotectant peptide with a molecular weight of 454.26 Da, and the amino acid sequence -NHGK showed remarkable thermal hysteresis activity (THA) of 2.63 °C. Frozen scallop muscles treated with 3 g/100g TSCP exhibited higher salt soluble protein concentration, total sulfhydryl content, Ca2+-ATPase activity, and water-holding capacity during the 8 weeks storage period. The cryoprotective effect of 3 g/100g TSCP was equivalent to that of the commercial phosphorus antifreeze. Our findings provide a theoretical basis for developing safe peptide-based cryoprotectants which will maintain and improve the quality of frozen foods.

Cover Feature: Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis (Chem. Eur. J. 21/2023)

The sugar moiety of the antifreeze glycoprotein AFGP can form a unique dynamic water phase that includes disordered water molecules thereby inhibiting the freezing process. The property of this water phase seems to depend on both the structure and the number of sugar moieties. More information can be found in the Research Article by R. Okamoto, Y. Kajihara, and co-workers (DOI: 10.1002/chem.202203553).

Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis.

Antifreeze glycoprotein (AFGP), which inhibits the freezing of water, is highly O-glycosylated with a disaccharide, d-Galβ1-3-d-GalNAcα (GalGalNAc). To elucidate the function of the sugar residues for antifreeze activity at the molecular level, we conducted a total chemical synthesis of partially sugar deleted AFGP derivatives, and unnatural forms of AFGPs incorporating glucose (Glc)-type sugars instead of galactose (Gal)-type sugars. These elaborated AFGP derivatives demonstrated that the stereochemistry of each sugar residue on AFGPs precisely correlates with the antifreeze activity. A hydrogen-deuterium exchange experiment using synthetic AFGPs revealed a different dynamic behavior of water around sugar residues depending on the sugar structures. These results indicate that sugar residues on AFGP form a unique dynamic water phase that disturbs the absorbance of water molecules onto the ice surface, thereby inhibiting freezing.

The Atomistic Understanding of the Ice Recrystallization Inhibition Activity of Antifreeze Glycoproteins

As the most potent ice recrystallization inhibitors, antifreeze glycoproteins (AFGPs) have been extensively studied since their discovery. However, the molecular mechanism of how they inhibit ice growth remains controversial—notably, which group directly contributes to the binding of AFGPs to ice is hotly debated. Here, we use molecular dynamics simulations to investigate the atomistic details of the binding of AFGP8 to ice. We show that the binding of AFGP8 to ice can be divided into three cases: backbone dominant binding (BDB), disaccharide dominant binding (DDB) and weak binding (WB). Hydrogen-bonding and hydrophobic groups contribute equally to the binding of AFGP8 to ice and synergistically promote the binding. The –CH3 groups promote the contacting of AFGP8 to ice via hydrophobic effect, and the hydrogen-bonding groups anchor AFGP8 to ice surfaces through direct hydrogen bonding with ice. Specially, we verify that the -CONH- groups anchor the backbone of AFGP8 to ice by forming hydrogen bonds with ice surfaces while the –OH groups not only anchor the disaccharide to ice but also slow down the dynamics of the surrounding water. In addition, we reveal that both the backbone and the disaccharide can bind to ice surfaces while the latter is more flexible, which also perturbs the hydrogen bond network of potential ice-like water molecules by swaying in the solution to further enhance its antifreeze activity. This work provides the atomistic details of the ice growth inhibition mechanism of AFGP8, which is helpful for the design of high-efficacy cryoprotectants.

Impact of meltwater flow intensity on the spatiotemporal heterogeneity of microbial mats in the McMurdo Dry Valleys, Antarctica

The meltwater streams of the McMurdo Dry Valleys are hot spots of biological diversity in the climate-sensitive polar desert landscape. Microbial mats, largely comprised of cyanobacteria, dominate the streams which flow for a brief window of time (~10 weeks) over the austral summer. These communities, critical to nutrient and carbon cycling, display previously uncharacterized patterns of rapid destabilization and recovery upon exposure to variable and physiologically detrimental conditions. Here, we characterize changes in biodiversity, transcriptional responses and activity of microbial mats in response to hydrological disturbance over spatiotemporal gradients. While diverse metabolic strategies persist between marginal mats and main channel mats, data collected from 4 time points during the austral summer revealed a homogenization of the mat communities during the mid-season peak meltwater flow, directly influencing the biogeochemical roles of this stream ecosystem. Gene expression pattern analyses identified strong functional sensitivities of nitrogen-fixing marginal mats to changes in hydrological activities. Stress response markers detailed the environmental challenges of each microhabitat and the molecular mechanisms underpinning survival in a polar desert ecosystem at the forefront of climate change. At mid and end points in the flow cycle, mobile genetic elements were upregulated across all mat types indicating high degrees of genome evolvability and transcriptional synchronies. Additionally, we identified novel antifreeze activity in the stream microbial mats indicating the presence of ice-binding proteins (IBPs). Cumulatively, these data provide a new view of active intra-stream diversity, biotic interactions and alterations in ecosystem function over a high-flow hydrological regime.

Cryoprotective effect of collagen hydrolysates from squid skin on frozen shrimp and characterizations of its antifreeze peptides

The cryoprotective effect of collagen hydrolysates from squid skin (CH-SS) on shrimp muscle was investigated during the freeze-thaw cycles, and the antifreeze peptides (AFPs) in CH-SS were separated and identified. The results showed that CH-SS generated by acid protease had the highest antifreeze activity. It was further found that CH-SS showed an inhibitory effect on the denaturation and structural changes of myofibrillar protein during the freeze-thaw cycles, and partially retained the ability to bind water. SEM analysis indicated that CH-SS could effectively reduce the mechanical injury caused by ice crystals to shrimp muscle. The fraction with the highest antifreeze activity from CH-SS was obtained by Sephadex G-25 chromatography, and its main amino acid sequence was identified as DVRGAEGSAGL by the UPLC-MS analysis. This study provides a theoretical basis for the intensive processing of squid skin and the development of new antifreeze agents.

Revealing the Mechanism of Irreversible Binding of Antifreeze Glycoproteins to Ice.

Antifreeze glycoproteins (AFGPs) are a special kind of antifreeze proteins with strong flexibility. Whether their antifreeze activity is achieved by reversibly or irreversibly binding to ice is widely debated, and the molecular mechanism of irreversible binding remains unclear. In this work, the antifreeze mechanism of the smallest AFGP isoform, AFGP8, is investigated at the atomic level. The results indicate that AFGP8 can bind to ice both reversibly through its hydrophobic methyl groups (peptide binding) and irreversibly through its hydrophilic disaccharide moieties (saccharide binding). Although peptide binding occurs faster than saccharide binding, free-energy calculations indicate that the latter is energetically more favorable. In saccharide binding, at least one disaccharide moiety is frozen in the grown ice, resulting in irreversible binding, while the other moieties significantly perturb the water hydrogen-bonding network, thus inhibiting ice growth more effectively. The present study reveals the coexistence of reversible and irreversible bindings of AFGP8, both contributing to the inhibition of ice growth and further provides molecular mechanism of irreversible binding.

Protein engineering of antifreeze proteins reveals that their activity scales with the area of the ice-binding site.

Antifreeze proteins (AFPs) protect organisms from freezing by binding to ice crystals to prevent their growth. Here, we have investigated how the area of an AFP's ice-binding site (IBS) changes its antifreeze activity. The polyproline type II helical bundle fold of the 9.6-kDa springtail (Collembola) AFP from Granisotoma rainieri (a primitive arthropod) facilitates changes to both IBS length and width. A one quarter decrease in area reduced activity to less than 10%. A one quarter increase in IBS width, through the addition of a single helix, tripled antifreeze activity. However, increasing IBS length by a similar amount actually reduced activity. Expanding the IBS area can greatly increase antifreeze activity but needs to be evaluated by experimentation on a case-by-case basis.

Molecular Factors of Ice Growth Inhibition for Hyperactive and Globular Antifreeze Proteins: Insights from Molecular Dynamics Simulation.

The molecular mechanism behind the ice growth inhibition by antifreeze proteins (AFPs) is yet to be understood completely. Also, what physical parameters differentiate between the AFP and non-AFP are largely unknown. Thus, to get an atomistic overview of the differential antifreeze activities of different classes of AFPs, we have studied ice growth from different ice surfaces in the presence of a moderately active globular type III AFP and a hyperactive spruce budworm (sbw) AFP. Results are compared with the observations of ice growth simulations in the presence of topologically similar non-AFPs using all-atom molecular dynamics simulations. Simulation data suggest that the ice surface coverage is a critical factor in ice growth inhibition. Due to the presence of an ice binding surface (IBS), AFPs form a high affinity complex with ice, accompanied by a transition of hydration water around the IBS from clathrate-like to ice-like. Several residues around the periphery of the IBS anchor the AFP to the curved ice surface mediated by multiple strong hydrogen bonds, stabilizing the complex immensely. In the high surface coverage regime, the slow unbinding kinetics dominates over the ice growth kinetics and thus facilitates the ice growth inhibition. Due to the non-availability of a proper IBS, non-AFPs form a low-affinity complex with the growing ice surface. As a result, the non-AFPs are continuously repelled by the surface. If the concentration of AFPs is low, then the effective surface coverage is reduced significantly. In this low surface coverage regime, AFPs can also behave like impurities and are engulfed by the growing ice crystal.

Identification of Novel Antifreeze Peptides from Takifugu obscurus Skin and Molecular Mechanism in Inhibiting Ice Crystal Growth.

Foodborne hydrolyzed antifreeze peptides have been widely used in the food industry and the biomedical field. However, the components of hydrolyzed peptides are complex and the molecular mechanism remains unclear. This study focused on identification and mechanism analysis of novel antifreeze peptides from Takifugu obscurus skin by traditional methods and computer-assisted techniques. Results showed that three peptides (EGPRAGGAPG, GDAGPSGPAGPTG, and GEAGPAGPAG) possessed cryoprotection via reducing the freezing point and inhibiting ice crystal growth. Molecular docking confirmed that the cryoprotective property was related to peptide structure, especially α-helix, and hydrogen bond sites. Moreover, the antifreeze peptides were double-faces, which controlled ice crystals while affecting the arrangement of surrounding water molecules, thus exhibiting a strong antifreeze activity. This investigation deepens the comprehension of the mechanism of antifreeze peptides at molecular scale, and the novel efficient antifreeze peptides can be developed in antifreeze materials design and applied in food industry.

Screening and characterization of a novel antifreeze peptide from silver carp muscle hydrolysate

This study aimed to screen and characterize antifreeze peptides from silver carp muscle hydrolysate (SCMH). The SCMH was initially fractionated by ultrafiltration, and the resultant SCMH-IV (<10 kDa) showing 90 % of yeast survival rate was subsequently separated into four fractions using ion-exchange chromatography. The fraction with the best antifreeze activity was further analyzed by liquid chromatography-tandem mass spectrometry. A total of 514 peptides were identified, of which a novel antifreeze peptide (Sc-AFP, KAADSFNHKAFFAKVG) with a thermal hysteresis activity of 0.87 ℃ was selected. The parvalbumin-derived Sc-AFP showed an alanine-rich, α-helical and amphipathic character. Based on molecular dynamics simulations, the Sc-AFP could interact with 48 water molecules via hydrogen bonds, and could be adsorbed onto the ice surface through a total of 21 hydrogen bonds mainly linked to the lysine residues. This could account for its antifreeze properties via preventing the formation and growth of ice crystals.

Molecular simulation -based research on antifreeze peptides: advances and perspectives

• Molecular simulation methods to study antifreeze peptides(AFPP) were summarized. • The structure-activity relationship of AFPP was analyzed from the perspective of amino acid composition and structure. • The factors and mechanisms affecting the antifreeze activity of AFPP by molecular simulation analysis were summarized. • The development trend of through molecular simulation to design antifreeze materials was prospected. Antifreeze protein (AFP) can inhibit the growth of ice crystals to protect organisms from freezing damage, and demonstrates broad application prospects in food industry. Antifreeze peptides (AFPP) are specific peptides with functional domains showing antifreeze activity in AFP. Bioinformatics-based molecular simulation technology can more accurately explain the properties and mechanisms of biological macromolecules. Therefore, the binding stability of antifreeze peptides and antifreeze proteins (AFP(P)) to ice and the molecular-scale growth kinetics of ice were analyzed by molecular simulation, which can make up for the limitations of experimental technology. This review concludes the molecular simulation-based research in the inhibition's study of AFP(P) on ice growth, including sequence prediction, structure construction, molecular docking and molecular dynamics (MD) studies of AFP(P) on ice applications in growth inhibition. Finally, the review prospects the future direction of designing new antifreeze biomimetic materials through molecular simulation and machine learning. The information presented in this paper will help enrich our understanding of AFPP.

Cold-inducible promoter-driven knockdown of Brachypodium antifreeze proteins confers freezing and phytopathogen susceptibility.

The model forage crop, Brachypodium distachyon , has a cluster of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were targeted for knockdown using a cold‐induced promoter from rice (prOsMYB1R35) to drive miRNA. The transgenic lines showed no apparent pleiotropic developmental defects but had reduced antifreeze activity as assessed by assays for ice‐recrystallization inhibition, thermal hysteresis, electrolyte leakage, and leaf infrared thermography. Strikingly, the number of cold‐acclimated transgenic plants that survived freezing at −8°C was reduced by half or killed entirely, depending on the line, compared with cold‐acclimated wild type plants. In addition, more leaf damage was apparent at subzero temperatures in knockdowns after infection with an ice nucleating pathogen, Pseudomonas syringae . Although antifreeze proteins have been studied for almost 60 years, this is the first unequivocal demonstration of their function by knockdown in any organism, and their dual contribution to freeze protection as well as pathogen susceptibility, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced pathogen and freezing tolerance.

A mutation to a fish ice-binding protein synthesized in transgenic Caenorhabditis elegans modulates its cold tolerance

A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at −5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.

Relationship between type II polyproline helix secondary structure and thermal hysteresis activity of short homopeptides

BackgroundAntifreezing activity is a phenomenon of great significance in food industry that affects the quality of frozen foods. As a solution, ice-binding proteins, more specifically antifreeze proteins, have been used to mitigate recrystallization. However, knowledge about the mechanism of ice recrystallization and the influence of antifreeze proteins is scarce. ResultsIn this work, model homopeptides of three amino acids (proline, arginine and lysine) were studied by means of differential scanning calorimetry through the determination of their thermal hysteresis activity, to see the influence of several factors on their secondary structure. It was found that model homopeptides formed polyproline II type secondary structure that was more stable at low temperature. In addition, thermal hysteresis activity was higher for peptides of intermediate lengths and for proline homopeptides. ConclusionsThe study of homopeptides sheds light on the mechanism of antifreeze activity and will allow the design of new molecules with antifreeze properties to be used in diverse biotechnological fields.How to cite: Rojas R, Aróstica M, Carvajal-Rondanelli P, et al. Relationship between type II polyproline helix secondary structure and thermal hysteresis activity of short homopeptides. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.08.003.

Multiple Adaptive Strategies of Himalayan Iodobacter sp. PCH194 to High-Altitude Stresses.

Bacterial adaption to the multiple stressed environments of high-altitude niches in the Himalayas is intriguing and is of considerable interest to biotechnologists. Previously, we studied the culturable and unculturable metagenome microbial diversity from glacial and kettle lakes in the Western Himalayas. In this study, we explored the adaptive strategies of a unique Himalayan eurypsychrophile Iodobacter sp. PCH194, which can synthesize polyhydroxybutyrate (PHB) and violacein pigment. Whole-genome sequencing and analysis of Iodobacter sp. PCH194 (4.58 Mb chromosome and three plasmids) revealed genetic traits associated with adaptive strategies for cold/freeze, nutritional fluctuation, defense against UV, acidic pH, and the kettle lake's competitive environment. Differential proteome analysis suggested the adaptive role of chaperones, ribonucleases, secretion systems, and antifreeze proteins under cold stress. Antifreeze activity inhibiting the ice recrystallization at −9°C demonstrated the bacterium's survival at subzero temperature. The bacterium stores carbon in the form of PHB under stress conditions responding to nutritional fluctuations. However, violacein pigment protects the cells from UV radiation. Concisely, genomic, proteomic, and physiological studies revealed the multiple adaptive strategies of Himalayan Iodobacter to survive the high-altitude stresses.

Comparative analysis of hydration layer reorientation dynamics of antifreeze protein and protein cytochrome P450

Antifreeze proteins (AFPs) inhibit ice re-crystallization by a mechanism remaining largely elusive. Dynamics of AFPs’ hydration water and its involvement in the antifreeze activity have not been identified conclusively. We herein, by simulation and theory, examined the water reorientation dynamics in the first hydration layer of an AFP from the spruce budworm, Choristoneura fumiferana, compared with a protein cytochrome P450 (CYP). The increase of potential acceptor water molecules around donor water molecules leads to the acceleration of hydrogen bond exchange between water molecules. Therefore, the jump reorientation of water molecules around the AFP active region is accelerated. Due to the mutual coupling and excitation of hydrogen bond exchange, with the acceleration of hydrogen bond exchange, the rearrangement of the hydrogen bond network and the frame reorientation of water are accelerated. Therefore, the water reorientation dynamics of AFP is faster than that of CYP. The results of this study provide a new physical image of antifreeze protein and a new understanding of the antifreeze mechanism of antifreeze proteins.

Cryoprotective effect of wheat gluten enzymatic hydrolysate on fermentation properties of frozen dough

This study investigated the antifreeze activities and structures of wheat gluten enzymatic hydrolysates at different degrees of hydrolysis and studied the cryoprotective effects of the hydrolysate with the highest antifreeze activity on fermentation properties of frozen dough. We found that the hydrolysate with an enzymolysis time of 90 min had the highest thermal hysteresis value (0.46 °C) and significantly inhibited growth and recrystallization of ice crystals. This might be due to the differences in molecular weight, amino acid composition, secondary structures, and surface hydrophobicity of the hydrolysates. Peptides with a molecular weight of 500–2000 Da accounted for 41.5% of the hydrolysate at 90 min, and this hydrolysate had greater levels of alanine, leucine, glycine, and glutamic acid residues, β-sheet conformation, and surface hydrophobicity than other hydrolysates. Furthermore, fermentation results indicated that the optimum condition for the highest active hydrolysates to protect frozen dough was 0.5% relative to the flour amount, and 0.5% hydrolysate enhanced the fermentation capacity of the frozen dough. Accordingly, wheat gluten enzymatic hydrolysate hydrolyzed for 90 min can protect dough from damage caused by repeated freezing–thawing. This study offers new insights into the preparation of antifreeze peptides and the use of wheat gluten.