Winter Flounder Antifreeze Protein Research Articles

Molecular insights into the synergistic inhibition mechanisms of antifreeze protein and methanol on carbon dioxide hydrate growth

The formation of CO2 hydrates during CO2 transportation and deep-sea injection poses a significant risk of pipeline blockage. Combining kinetic (KHIs) and thermodynamic hydrate inhibitors (THIs) serves as a promising efficient and economical strategy to mitigate this issue, whose performance and microscopic mechanisms yet are still poorly understood. This study employs molecular dynamics simulations to explore the effects of the combination of winter flounder antifreeze protein (wf-AFP) as a KHI and methanol as a THI on CO2 hydrate growth. We find that methanol and wf-AFP exhibit an intriguing synergistic inhibition performance on hydrate growth. In the AFP-only system, AFP serves as a spatial hindrance near the hydrate growth interface. However, in the AFP + methanol system, AFP changes its position due to the attractive force of methanol. Part of the AFP fragment remains on the hydrate interface, while the rest surrounds the CO2 droplet. Methanol, in addition to disrupting the water structure, combines with AFP to act as a double barrier to CO2 dissolution into the aqueous solution, thereby significantly reducing the gas source for hydrate growth. These findings provide molecular-level insights into hydrate inhibition mechanisms and guide the design of efficient inhibitors for safe CO2 transportation and injection.

The effect of winter flounder antifreeze protein and its mutants on methane hydrate growth: A molecular dynamic simulation study

The effect of winter flounder antifreeze protein and its mutants on methane hydrate growth: A molecular dynamic simulation study

Paradoxical effects on ice nucleation are intrinsic to a small winter flounder antifreeze protein

Antifreeze proteins (AFPs) bind to ice in solutions, resulting in non-colligative freezing point depression; however, their effects on ice nucleation are not well understood. The predominant plasma AFP of winter flounder (Pseudopleuronectes americanus) is AFP6, which is an amphiphilic alpha helix. In this study, AFP6 and modified constructs were produced as fusion proteins in Escherichia coli, subjected to proteolysis when required and purified prior to use. AFP6 and its recombinant fusion precursor generated similar thermal hysteresis and bipyramidal ice crystals, whereas an inactive mutant AFP6 produced hexagonal crystals and no hysteresis. Circular dichroism spectra of the wild-type and mutant AFP6 were consistent with an alpha helix. The effects of these proteins on ice nucleation were investigated alongside non-AFP proteins using a standard droplet freezing assay. In the presence of nucleating AgI, modest reductions in the nucleation temperature occurred with the addition of mutant AFP6, and several non-AFPs, suggesting non-specific inhibition of AgI-induced ice nucleation. In these experiments, both AFP6 and its recombinant precursor resulted in lower nucleation temperatures, consistent with an additional inhibitory effect. Conversely, in the absence of AgI, AFP6 induced ice nucleation, with no other proteins showing this effect. Nucleation by AFP6 was dose-dependent, reaching a maximum at 1.5 mM protein. Nucleation by AFP6 also required an ice-binding site, as the inactive mutant had no effect. Furthermore, the absence of nucleation by the recombinant precursor protein suggested that the fusion moiety was interfering with the formation of a surface capable of nucleating ice.

De novo designed ice-binding proteins from twist-constrained helices

Attaining molecular-level control over solidification processes is a crucial aspect of materials science. To control ice formation, organisms have evolved bewildering arrays of ice-binding proteins (IBPs), but these have poorly understood structure-activity relationships. We propose that reverse engineering using de novo computational protein design can shed light on structure-activity relationships of IBPs. We hypothesized that the model alpha-helical winter flounder antifreeze protein uses an unusual undertwisting of its alpha-helix to align its putative ice-binding threonine residues in exactly the same direction. We test this hypothesis by designing a series of straight three-helix bundles with an ice-binding helix projecting threonines and two supporting helices constraining the twist of the ice-binding helix. Our findings show that ice-recrystallization inhibition by the designed proteins increases with the degree of designed undertwisting, thus validating our hypothesis, and opening up avenues for the computational design of IBPs.

Investigation on structural properties of winter flounder antifreeze protein in interaction with clathrate hydrate by molecular dynamics simulation

Antifreeze proteins (AFPs) are a kind of biomolecules that allow organisms surviving in subzero environments and have been applied as kinetic hydrate inhibitors recently. It was shown that some residues of type I AFP bind to the hydrate cages and consequently inhibit hydrate growth. However, the exact amino acids which adsorb to the hydrate surface has not yet elucidated. In this study, the AFP I inhibition pattern on hydrate formation has been investigated. It was found that amino acids played a significant role in hydrate binding. Different structural and dynamical parameters on protein residues and water molecules at the hydrate demonstrated this hydrate binding site (HBS) of AFP. It was found that AFP I adsorbs to the hydrate surface from Thr2 or Thr35 and bends around residue 15–17 as a result of hydrate binding. It was proposed that Thr-(i), Ala-(i ± 4), Ala-(i ± 7) and Ala-(i ± 18) provide a good shape complementary to replace on the half-cages of hydrate surface instead of guest molecules. Therefore, adsorption of AFP I from Thr2 in IBS drives the methyl groups of Ala6, Ala18, and Ala20 to entrap in hydrate cages at the interface, while the methyl group of Ala31, Ala28, and Ala17 place in the empty half-cages of the hydrate when adsorbed from terminal Thr35.

The Inhibition of Icing and Frosting on Glass Surfaces by the Coating of Polyethylene Glycol and Polypeptide Mimicking Antifreeze Protein.

The development of anti-icing, anti-frosting transparent plates is important for many reasons, such as poor visibility through the ice-covered windshields of vehicles. We have fabricated new glass surfaces coated with polypeptides which mimic a part of winter flounder antifreeze protein. We adopted glutaraldehyde and polyethylene glycol as linkers between these polypeptides and silane coupling agents applied to the glass surfaces. We have measured the contact angle, the temperature of water droplets on the cooling surfaces, and the frost weight. In addition, we have conducted surface roughness observation and surface elemental analysis. It was found that peaks in the height profile, obtained with the atomic force microscope for the polypeptide-coated surface with polyethylene glycol, were much higher than those for the surface without the polypeptide. This shows the adhesion of many polypeptide aggregates to the polyethylene glycol locally. The average supercooling temperature of the droplet for the polypeptide-coated surface with the polyethylene glycol was lower than for the polypeptide-coated surface with glutaraldehyde and the polyethylene-glycol-coated surface without the polypeptide. In addition, the average weight of frost cover on the specimen was lowest for the polypeptide-coated surface with the polyethylene glycol. These results argue for the effects of combined polyethylene glycol and polypeptide aggregates on the locations of ice nuclei and condensation droplets. Thus, this polypeptide-coating with the polyethylene glycol is a potential contender to improve the anti-icing and anti-frosting of glasses.

Effects of Winter Flounder Antifreeze Protein on the Growth of Ice Particles in an Ice Slurry Flow in 
 Mini-Channels

The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of the promising methods for the control is to use a substance such as antifreeze protein. We have observed and report here growth states of ice particles in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in mini-channels with a microscope. We also measured ice growth rates. Our aim was to improve the levels of ice growth inhibition by subjecting the antifreeze protein solution both to preheating and to concentrating by ultrafiltration. We have found that the ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. In addition, unlike unidirectional freezing experiments, the preheating of the antifreeze protein solution reduced the ice growth inhibition properties. This is because the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, and thus the probability of interaction between HPLC6 and ice surfaces does not increase. In contrast to this, ultrafiltration after preheating the solution improved the ice growth inhibition. This may be due to the interaction between ice surfaces and many aggregates in the concentrates.

Suppression of droplets freezing on glass surfaces on which antifreeze polypeptides are adhered by a silane coupling agent.

The development of ice-phobic, glass-substrate surfaces is important for many reasons such as poor visibility through the ice-covered windshields of vehicles. The present authors have developed new glass surfaces coated with a silane coupling agent and polypeptides whose amino-acid sequence is identical to a partial sequence of winter flounder antifreeze protein. We have conducted experiments on the freezing of sessile water droplets on the glass surfaces, and measured the droplet temperature, contact angle, contact area and surface roughness. The results show that the supercooling temperature decreased noticeably in the case where a higher concentration solution of polypeptide was used for the coating. The adhesion strength of frozen droplets was lowest in the same case. In addition, we observed many nanoscale humps on the coated surface, which were formed by polypeptide aggregates in the solution. We argue that the combination of the hydrophilic humps and the hydrophobic base surfaces causes water molecules adjacent to the surfaces to have a variety of orientations in that plane, even after the ice layer started to grow. This then induces a misfit of water-molecule spacing in the ice layers and consequent formation of fragile polycrystalline structure. This explains the lower values of ice adhesion strength and supercooling enhancement in the cases of the polypeptide-coated glass plates.

Role of Polar and Nonpolar Groups in the Activity of Antifreeze Proteins: A Molecular Dynamics Simulation Study

Molecular dynamics simulations have been carried out separately with the hyperactive Tenebrio molitor antifreeze protein ( TmAFP) and with its nonactive mutant at 300 K to elucidate the role of polar and nonpolar groups in the activities of antifreeze proteins (AFPs). Simulation results reveal that both polar and nonpolar groups contribute to develop the required quasi-ice-like hydration layer on the ice-binding surface (IBS) of an AFP for binding onto ice. Nonpolar groups on the IBS induce the formation of locally ordered icelike low-density waters in the hydration layer through hydrophobic interactions, and polar groups of the surface integrate these waters into a quasi-ice-like layered structure through hydrogen-bonding interactions. These contributions of polar and nonpolar groups apparently contradict the behavior of winter flounder antifreeze protein (wfAFP) mutants possibly due to switching of IBS of wfAFP upon mutation of threonine residues with valine residues.

Molecular Dynamics Analysis of Synergistic Effects of Ions and Winter Flounder Antifreeze Protein Adjacent to Ice-Solution Surfaces

The control of freezing saline water at the micrometer level has become very important in cryosurgery and cryopreservation of stem cells and foods. Adding antifreeze protein to saline water is a promising method for controlling the freezing because the protein produces a gap between the melting point and the freezing point. Furthermore, a synergistic effect of the solutes occurs in which the freezing point depression of a mixed solution is more noticeable than the sum of two freezing point depressions of single-solute solutions. However, the mechanism of this effect has not yet been clarified. Thus, we have carried out a molecular dynamics simulation on aqueous solutions of winter flounder antifreeze protein and sodium chloride or calcium chloride with an ice layer. The results show that the cations inhibit the hydrogen bond among water molecules not only in the salt solutions but also in the mixed solutions. This inhibition depends on the local number of ions and the valence of cations. The space for water molecules to form the hydrogen bonds becomes small in the case of the mixed solution of the protein and calcium chloride. These findings are consistent with the synergistic effect. In addition, it is found that the diffusion of ions near positively-charged residues is attenuated. This attenuation causes an increase in the possibility of water molecules staying near or inside the hydration shells of the ions. Furthermore, the first hydration shells of the cations become weak in the vicinity of the arginine, lysine and glutamic-acid residues. These factors can be considered to be possible mechanisms of the synergistic effect.

Effects of short-time preheating on ice growth in antifreeze polypeptides solutions in a narrow space

We conducted experiments on the unidirectional freezing of solutions of winter flounder antifreeze protein or of a polypeptide which was based on twelve amino-acid residues of this protein. The temperature in the solutions and ice was measured with a small thermocouple. The interface temperature was defined as the temperature at the tip of the serrated or pectinate interface. The interface temperature of these solutions was lower than that of pure water. To vary this supercooling activity of these solutes, we preheated the solutions and cooled them before conducting identical experiments. It was found that short-time preheating caused further decreases in the interface temperature and interface velocities. Furthermore, the inclined interfaces and the narrow liquid regions inside the ice area became wider. To investigate the reasons for these changes, we measured aggregates of the solutes in the solutions. These aggregates were found to become larger as a result of preheating. Thus, it can be concluded that these large aggregates attenuated the ice growth by their interaction with ice. Finally, we carried out similar measurements by using pH-adjusted solutions of the protein to produce aggregates without preheating, and obtained similar supercooling enhancement by the aggregates. Thus, the effects of thermal denaturation on the supercooling were not significant in the preheating.

Effects of preheating on ice growth in antifreeze polypeptides solutions in a narrow space

We conducted measurements on the unidirectional freezing of aqueous solutions of polypeptide or of winter flounder antifreeze protein. The polypeptide was based on a part of the antifreeze protein. We measured temperatures in the solutions and ice with a small thermocouple, and defined the interface temperature as the temperature at the tip of the serrated or pectinate interface. It was found that the interface temperature of these solutions was lower than that of pure water. To vary the activity of these solutes, we preheated the solutions and cooled them before conducting the measurements. We found that preheating for several hours caused further decreases in the interface temperature and a decrease in the interface velocity. In addition, the inclined interfaces became wider as a result of the preheating. Thus, the supercooled states in the solutions were enhanced by the preheating. To investigate the reasons for these changes, we measured the aggregates of the solutes in the solutions. These aggregates became larger as a result of preheating. It can therefore be concluded that these large aggregates attenuated the ice growth by their interaction with the ice surfaces.

Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating.

The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure.

M1008 冬ガレイ由来不凍タンパク質の一部を基にしたポリペプチドの氷成長抑制に関する分子動力学解析(GS6 熱工学(2),修士研究発表セッション)

M1008 冬ガレイ由来不凍タンパク質の一部を基にしたポリペプチドの氷成長抑制に関する分子動力学解析(GS6 熱工学(2),修士研究発表セッション)

分子動力学解析を用いた冬ガレイ由来不凍タンパク質とイオンが有する水分子の運動抑制効果の調査

We have carried out molecular dynamics analysis for water with an ice wall, winter flounder antifreeze protein and ions to understand mechanism of a ‘synergistic effect’. This effect shows the freezing point of mixed solution is lower than the sum of the freezing point of antifreeze protein solution and the freezing point of salt solution. It is found that movement of water molecules is more inhibited by the interaction of antifreeze protein and ions than the movement of water moleculars in each single-solute solution. This may be due to the fact that the antifreeze protein strengthens hydration shell around sodium ions.

二種類のポリペプチド水溶液の氷表面温度と円偏光二色性の計測

We conducted measurements on unidirectional freezing and circular dichroism for preheated solution and unheated solution of a polypeptide based on winter flounder antifreeze protein. The interface temperature were measured with a thermocouple. The secondary structure of this polypeptide was measured with circular dichroism spectrophotometer. It was found from the circular dichroism spectra that the polypeptide had a β-sheet structure. Although the interface temperature for the unheated solution of the polypeptide did not drop, the interface temperature for the preheated solution showed significant dropped.

Will It Be Beneficial To Simulate the Antifreeze Proteins at Ice Freezing Condition or at Lower Temperature?

Antifreeze proteins (AFPs) enable the polar living species to survive subzero temperature conditions through effective lowering of the freezing point of body fluids. At the molecular level, AFPs directly interact with the growing seeds of ice crystals to inhibit their formation. To understand the structural and dynamic aspects of this interaction at the atomistic level, molecular dynamics (MD) simulations were carried out on several type I AFPs at multiple temperatures, including the physiologically relevant temperature of 273 K, a lower temperature of 227 K, and the conventional 300 K. A comparison of the principal component analysis (PCA) and mean squared deviation plots for Winter flounder AFP, HPLC6 (mutant of winter flounder AFP), Sculpin, and peptide 1m AFPs reveals that simulations at 273 and 227 K result in the formation of more conserved metastable states than at 300 K. Other parameters such as root-mean-square deviation (rmsd), solvent accessibility surface area (SASA), H-bonding and residual density function (RDF) also suggest the same. MD simulations with ice crystal, where AFPs are complexed to ice plane with TIP4P/ice water model, help in finding relevance of dynamic behavior, and physiological temperature becomes more pronounced. Additionally, a control study on a nonantifreeze protein (LL37) is included, which aids in exploring significant information. On the basis of this approach, it was found that AFPs at 273 and 227 K display relevant dynamic properties that appear at 300 K for nonantifreeze proteins. The present study hence emphasizes the importance of performing computational simulations for antifreeze proteins at the physiologically relevant temperature (273 K), and even at lower temperatures (like 227 K), rather than at room temperatures (300 K).

Supercooling enhancement by adding antifreeze protein and ions to water in a narrow space

The control of ice crystal growth has been an important issue in various fields, such as the quality maintenance and improvement of food texture in frozen food, and the storages of tissues and organs in hospitals. The adding of antifreeze protein to food or organs has been focused on recently because it is appropriate for controlling ice growth. However, the effects of ions, which are originally contained in the substances being preserved, on the activities of antifreeze protein have not yet been clarified in a thermal non-equilibrium state. We therefore conducted experiments on the unidirectional freezing of dilute aqueous solutions of winter flounder antifreeze protein, sodium chloride or sodium permanganate in a narrow space between two cover glasses. The temperatures at ice/solution interfaces were measured with a small thermocouple. The concentration of antifreeze protein was estimated by fluorescence microscopy, and the concentration of permanganate ion was measured from the intensity of transmitted light. It was found that the depression of interface temperature (defined as the difference between the interface temperature of the solution and that of pure water) of the mixed solution was much lower than the sum of the depression of the interface temperatures of two solutions of single solute. In addition, we found that the protein concentration increases with time, while the ion concentration decreases slightly in the front regions of the most advanced points of serrated interfaces in the mixed solution. The diffusion of ions to the ice growth direction enhances the protein diffusion to the front-edge regions. This is possibly the reason for the low value of the interface temperature of the mixed solution. Simultaneously, high concentration regions of the protein in the bottom-edge region deteriorate the diffusion of ions. This is reason for the deterioration of the depression of interface temperature due to an increase in the interface velocity in the cases of mixed solutions.

Why ice-binding type I antifreeze protein acts as a gas hydrate crystal inhibitor.

Antifreeze proteins (AFPs) prevent ice growth by binding to a specific ice plane. Some AFPs have been found to inhibit the formation of gas hydrates which are a serious safety and operational challenge for the oil and gas industry. Molecular dynamics simulations are used to determine the mechanism of action of the winter flounder AFP (wf-AFP) in inhibiting methane hydrate growth. The wf-AFP adsorbs onto the methane hydrate surface via cooperative binding of a set of hydrophobic methyl pendant groups to the empty half-cages at the hydrate/water interface. Each binding set is composed of the methyl side chain of threonine and two alanine residues, four and seven places further down in the sequence of the protein. Understanding the principle of action of AFPs can lead to the rational design of green hydrate inhibitor molecules with potential superior performance.

G133 不凍タンパク質を基にしたポリペプチドを固定した面における氷成長の抑制(OS-14:凝固・融解伝熱および結晶成長の新展開(3))

The development of icephobic surfaces is an important issue in recent years. We have created a new functional surface for lowering the freezing temperature of a water droplet by adhering polypeptides on the surface. These polypeptides are based on the part of winter flounder antifreeze protein. To examine the effectiveness of the surface, we conducted simultaneous measurements of water droplet temperature on the cooling surface with a fine thermocouple and solidification of the droplet with a microscope. Although there is no change in the coagulation of water droplet, we obtained the depression of freezing temperature due to the surface.