Plane Of Ice Research Articles

Can clathrates heterogeneously nucleate ice?

Methane hydrates can be preserved at ambient pressure, beyond their region of thermodynamic stability, by storing them at temperatures from 240 to 270 K. The origin of this anomalous self-preservation is the formation of an ice coating that covers the clathrate particles and prevents further loss of gas. While there have been several studies on self-preservation, the question of what is the mechanism by which ice nucleates on the decomposing clathrate hydrates has not yet been fully explained. Here, we use molecular simulations, thermodynamic analysis, and nucleation theory to investigate possible scenarios for the nucleation of ice: heterogeneous nucleation at the clathrate/vapor or clathrate/liquid interfaces and homogeneous nucleation from supercooled water. Our results indicate that clathrates cannot heterogeneously nucleate ice and that ice nucleation is due to the cooling of water at the decomposing clathrate/liquid interface, which suffices to trigger homogeneous ice nucleation. We find that the (111) face of the sII structure clathrate can bind to the (111) plane of cubic ice or the basal plane of hexagonal ice through domain matching, resulting in a weak binding that-while insufficient to promote heterogeneous ice nucleation-suffices to produce epitaxy and alignment between these crystals. We use thermodynamic relations, theory, and the contact angles of ice at the (111) sII clathrate/liquid interface to determine-for the first time-the interfacial free energy of this most favorable ice-clathrate interface, 59 ± 5 mJ/m2. We discuss the implications of our results for the feasibility of heterogeneous nucleation of gas clathrates at ice/vapor interfaces.

Calcium ion implicitly modulates the adsorption ability of ion-dependent type II antifreeze proteins on an ice/water interface: a structural insight.

Ion dependent type II antifreeze proteins (AFPs) are an unusual design of natural evolution for cold-acclimatization of fishes in the Antarctic region. This class of proteins requires Ca2+ to perform an unusual biological recognition, binding to a specific ice plane. However, an ice-protein complex is yet to be characterized at the molecular scale. Here, using equilibrium simulations, free energy calculations and metadynamics, we have elucidated this unusual ice recognition phenomenon at the atomistic level. The origin of ion selectivity has been critically investigated to identify the role of different ions in the dynamics and ice binding ability of the protein. We have demonstrated that within the type II protein matrix, the preferred coordination number of Ca2+ is seven involving five protein atoms and two water molecules. Due to this coordination geometry, the ion binding loop adopts a flat solvent exposed conformation which helps the AFP to efficiently adsorb on the prism plane. The ice binding surface (IBS) adsorbs on the ice surface mediated by a layer of ordered water. Structural synergy between the ice/water interface of the prism plane and the water structure around the IBS makes the adsorption highly favorable. On the other hand, the preferred geometry of the Zn2+ coordination sphere within the AFP matrix is tetrahedral. Both the coordination number and the coordination bond length are smaller for Zn2+ in comparison to Ca2+. Thus to optimize the coordination sphere for Zn2+ within the protein matrix, a kink is introduced in the ion binding loop, a part of the IBS. Therefore, the IBS and ice surface complementarity is greatly perturbed which leads to less effective adsorption.

Investigation of Vibration Induced by Sliding Down an Ice Plane

Many studies use an inclined ice plane to determine the effect of different materials and processing conditions on sliding over an ice surface. Experiments measure the sliding time at different stages on the track to determine the sliding speed and the coefficient-of-friction. Here, the sliding body vibrations and, the oscillations in axial directions are addressed. This paper analyzes the sample’s acceleration by attaching a portable High Sensitivity 1.25g USB Accelerometer and compares the data with 3D CAM modeling results to identify the causes for interrupted sliding.

Ice recrystallization is strongly inhibited when antifreeze proteins bind to multiple ice planes

Ice recrystallization is a phenomenon observed as the increase in ice crystal size within an already frozen material. Antifreeze proteins (AFPs), a class of proteins capable of arresting ice crystal growth, are known to inhibit this phenomenon even at sub milli-molar concentrations. A tremendous range in the possible applications of AFPs is hence expected in both medical and industrial fields, while a key determinant of the ice recrystallization inhibition (IRI) is hardly understood. Here, IRI efficiency and ice plane affinity were examined for the wild-type AFPI–III, a defective AFPIII isoform, and a fungal AFP isoform. To simplify the IRI analysis using the formal representation of Ostwald-ripening (r3 = r03 + kt), we monitored specific ice grains exhibiting only uniform growth, for which maximum Feret diameter was measured. The cube of an ice grain’s radius (r3) increased proportionately with time (t), and its slope gave the recrystallization rate (k). There was a significant difference in the IRI efficiency between the samples, and the fungal AFP possessing the activity with the smallest amount (0.27 μM) exhibited an affinity to multiple ice planes. These results suggest that the IRI efficiency is maximized when AFPs bind to a whole set of ice planes.

Comparative Adsorption of Acetone on Water and Ice Surfaces.

Small organic molecules on ice and water surfaces are ubiquitous in nature and play a crucial role in many environmentally relevant processes. Herein, we combine surface‐specific vibrational spectroscopy and a controllable flow cell apparatus to investigate the molecular adsorption of acetone onto the basal plane of single‐crystalline hexagonal ice with a large surface area. By comparing the adsorption of acetone on the ice/air and the water/air interface, we observed two different types of acetone adsorption, as apparent from the different responses of both the free O−H and the hydrogen‐bonded network vibrations for ice and liquid water. Adsorption on ice occurs preferentially through interactions with the free OH group, while the interaction of acetone with the surface of liquid water appears less specific.

Evaluation of Thermal Hysteresis Activity of Ice-Binding Protein using Molecular Dynamics Simulation

Ice-etching experiment and molecular docking demonstrated that two ice-binding proteins (IBP), LeIBP from artic yeast and FfIBP from sea ice bacterium, show different activities in suppressing the growth of ice, leading to different thermal hysteresis (TH) even though they share common features in primary/tertiary structures. In this study, we investigated the ice-binding pattern of the two IBPs to different ice planes, prism and basal plane using all-atom molecular dynamics simulation. Our results provide a fully atomistic description of the difference in TH of two IBPs.

Point Mutation of the Ice-Binding Site in Antifreeze Protein Modify the Cold Tolerance in Caenorhabditis Elegans

Antifreeze proteins (AFPs) inhibit the ice-crystal growth from the surface-bound waters that are spatially organized to bind to an ice crystal. The surface structure complementarity between AFP and the ice plane has been evaluated based on structural docking studies. The ice-binding properties are closely related to the freezing point depression activity of the AFP solution, which is evaluated as the difference between the melting and freezing temperatures (thermal hysteresis: TH). Although AFPs are thought to facilitate the survival of organisms living in cold environments, it is poorly understood that whether the exogenous expression of AFPs can improve the cold tolerance of animals. Moreover, it is also unclear whether TH activity is correlated with the survival ability of animals during cold shock. Here, we examined the functions of AFPs in vivo through behavioral and cellular observations of transgenic Caenorhabditis elegans expressing AFPs. We showed that the fungal AnpAFP improves the survival rate of worms and protects their cells from lethal cold shocks. The AnpAFP T156Y mutant exhibiting low TH resulted the reduced cold tolerance at the freezing temperature. The fungal TisAFP8 exhibiting high TH activity was observed to dramatically improve the survival rate and strongly protect muscle cells. Structural modification of the ice-binding site is crucial to regulate the ice-binding strength of AFPs. These results suggest that AFPs play a crucial role in the animal body, improving cold tolerance through the ice-binding mechanism. In this presentation, we will discuss the relationship between the ice-binding abilities of AFPs and their in vivo roles in cold tolerance and cellular protection in a living animal.

Elucidating the role of key structural motifs in antifreeze glycoproteins.

Antifreeze glycoproteins (AFGPs) are distinctively riveting class of bio-macromolecules, which endows the survival of organisms inhabiting polar and subpolar regions. These proteins are believed to hinder microscopic freezing by interacting with embryonic ice crystals and precluding their further growth. The underlying molecular mechanism by which AFGPs bind to ice has remained elusive due to insufficient structural characterization, with conflicting hypotheses on the possible binding mode of AFGPs - either via the hydrophobic peptide backbone or via the hydrophilic carbohydrate side chains - when interacting with ice. Chemical synthesis has allowed researchers to access synthetic variants of natural AFGPs. These studies revealed that AFGPs exhibit huge variations in their thermal hysteresis and ice shaping behavior with only slight structural variations, especially to the carbohydrate side chains. Four key structural motifs were identified as crucial to AFGP activity: the presence of a threonine γ-methyl group, an α-glycosidic carbohydrate-protein linkage, an acetylamide group (-NHCOCH3) at the C2 position of the carbohydrate linked to the protein, and the presence of carbohydrate hydroxyl groups. In this study, we use molecular dynamics (MD) simulations to probe the microscopic properties of water accompanying these structural variations of AFGPs. We find that these variations primarily influence the conformation space of AFGPs and also crucially control their hydration dynamics. Owing to the disordered nature of AFGPs we use Markov-state modeling to identify the conformational preferences of AFGPs. The simulations reveal the importance of steric bulk, intra-molecular carbohydrate-protein H-bonds and conformational preferences (α- vs. β-linkages) in controlling the spatial segregation of the hydrophilic and hydrophobic regions of AFGPs. We hypothesize that the hydrophobic component of AFGPs is crucial to their binding to ice, which determines the ice shaping ability of AFGPs. However, the hydrophilic carbohydrate hydroxyl groups and their ability to form water bridges control the subsequent hydration dynamics, which is key to the antifreeze properties. Investigating the tetrahedral order parameter of water molecules around the carbohydrates revealed competition between solute- and bulk-influenced solvent structures, with maximum restructuring being observed in the interfacial region 2.5-4.5 Å away from the AFGPs.

Effects of hydrophobic and hydrogen-bond interactions on the binding affinity of antifreeze proteins to specific ice planes

Tenebrio molitor antifreeze protein (TmAFP) was simulated with growing ice surfaces such as primary prism, secondary prism, basal, and pyramidal planes. The ice-binding site of TmAFP, which is full of threonine (Thr), binds to the primary-prism plane but does not bind to other ice planes, in agreement with experiments showing the fast adsorption of TmAFP to the primary-prism plane. To mimic the ice-binding site of shorthorn sculpin AFP (ssAFP; type I) that predominantly consists of alanine (Ala) and has the binding affinity to the secondary-prism plane, the ice-binding site of TmAFP was mutated by replacing a few Thr residues with Ala residues, showing that mutated TmAFP binds to the secondary-prism plane, similar to the ice-binding affinity of ssAFP. Ala residues are located at the cavity of ice, while Thr residues form hydrogen bonds with water molecules. When the mutated TmAFP is further modified by removing Thr, it does not bind to the secondary-prism plane. These findings indicate that simulations can successfully capture the experimentally observed binding affinity of AFP to specific ice planes, to an extent dependent on hydrophobicity of the ice-binding site. In particular, the addition of hydrophobic residues influences the ice-binding affinity of TmAFP, while a certain amount of hydrophilic residue is still required for hydrogen-bond interactions, which supports experimental observations regarding the key roles of hydrophobic and hydrophilic interactions on the AFP-ice binding.

Saturn-Shaped Ice Burst Pattern and Fast Basal Binding of an Ice-Binding Protein from an Antarctic Bacterial Consortium.

Ice-binding proteins (IBPs) bind to ice crystals and control their growth, enabling host organisms to adapt to subzero temperatures. By binding to ice, IBPs can affect the shape and recrystallization of ice crystals. The shapes of ice crystals produced by IBPs vary and are partially due to which ice planes the IBPs are bound to. Previously, we have described a bacterial IBP found in the metagenome of the symbionts of Euplotes focardii ( EfcIBP). EfcIBP shows remarkable ice recrystallization inhibition activity. As recrystallization inhibition of IBPs and other materials are important to the cryopreservation of cells and tissues, we speculate that the EfcIBP can play a future role as an ice recrystallization inhibitor in cryopreservation applications. Here we show that EfcIBP results in a Saturn-shaped ice burst pattern, which may be due to the unique ice-plane affinity of the protein that we elucidated using the fluorescent-based ice-plane affinity analysis. EfcIBP binds to ice at a speed similar to that of other moderate IBPs (5 ± 2 mM-1 s-1); however, it is unique in that it binds to the basal and previously unobserved pyramidal near-basal planes, while other moderate IBPs typically bind to the prism and pyramidal planes and not basal or near-basal planes. These insights into EfcIBP allow a better understanding of the recrystallization inhibition for this unique protein.

Multivalent Presentation of Ice Recrystallization Inhibiting Polymers on Nanoparticles Retains Activity.

Poly(vinyl alcohol) (PVA) has emerged as the most potent mimic of antifreeze (glyco)proteins ice recrystallization inhibition (IRI) activity, despite its lack of structural similarities and flexible, rather than rigid, backbone. The precise spacing of hydroxyl groups is hypothesized to enable PVA to recognize the prism planes of ice but not the basal plane, due to hydroxyl pattern matching of the ice surface giving rise to the macroscopic activity. Here, well-defined PVA derived from reversible addition–fragmentation chain-transfer (RAFT) polymerization is immobilized onto gold nanoparticles to enable the impact of nanoscale assembly and confinement on the observed IRI activity. Unlike previous reports using star-branched or bottle-brush PVAs, the nanoparticle–PVA retains all IRI activity compared to polymers in solution. Evidence is presented to show that this is due to the low grafting densities on the particle surface meaning the chains are free to explore the ice faces, rather than being constrained as in star-branched polymers. These results demonstrate a route to develop more functional IRI’s and inclusion of metallic particle cores for imaging and associated applications in cryobiology.

Polypentagonal ice-like water networks emerge solely in an activity-improved variant of ice-binding protein

Polypentagonal water networks were recently observed in a protein capable of binding to ice crystals, or ice-binding protein (IBP). To examine such water networks and clarify their role in ice-binding, we determined X-ray crystal structures of a 65-residue defective isoform of a Zoarcidae-derived IBP (wild type, WT) and its five single mutants (A20L, A20G, A20T, A20V, and A20I). Polypentagonal water networks composed of ∼50 semiclathrate waters were observed solely on the strongest A20I mutant, which appeared to include a tetrahedral water cluster exhibiting a perfect position match to the [Formula: see text] first prism plane of a single ice crystal. Inclusion of another symmetrical water cluster in the polypentagonal network showed a perfect complementarity to the waters constructing the [Formula: see text] pyramidal ice plane. The order of ice-binding strength was A20L < A20G < WT < A20T < A20V < A20I, where the top three mutants capable of binding to the first prism and the pyramidal ice planes commonly contained a bifurcated γ-CH3 group. These results suggest that a fine-tuning of the surface of Zoarcidae-derived IBP assisted by a side-chain group regulates the holding property of its polypentagonal water network, the function of which is to freeze the host protein to specific ice planes.

An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin.

Submitted new structure to the Protein Data Bank (PDB: 6BG8).

Optimum Number of Anchored Clathrate Water and Its Instantaneous Fluctuations Dictate Ice Plane Recognition Specificities of Insect Antifreeze Protein.

Ice recognition by antifreeze proteins (AFPs) is a subject of topical interest. Among several classes of AFPs, insect AFPs are hyperactive presumably due to their ability to adsorb on basal plane. However, the origin of the basal plane binding specificity is not clearly known. Present work aims to provide atomistic insight into the origin of basal plane recognition by an insect antifreeze protein. Free energy calculations reveal that the order of binding affinity of the AFP toward different ice planes is basal plane > prism plane > pyramidal plane. Critical insight reveals that the observed plane specificity is strongly correlated with the number and their instantaneous fluctuations of clathrate water forming hydrogen bonds with both ice binding surface (IBS) of AFP and ice surface, thus anchoring AFP to the ice surface. On basal plane, anchored clathrate water array is highly stable due to exact match in the periodicity of oxygen atom repeat distances of the ice surface and the threonine repeat distances at the IBS. The stability of anchored clathrate water array progressively decreases upon prism and pyramidal plane adsorption due to mismatch between the threonine ladder and oxygen atom repeat distance. Further analysis reveals that hydration around the methyl side-chains of threonine residues becomes highly significant at low temperature which stabilizes the anchored clathrate water array and dual hydrogen-bonding is a consequence of this stability. Structural insight gained from this study paves the way for rational designing of highly potent antifreeze-mimetic with potential industrial applications.

Adsorption of Kinetic Hydrate Inhibitors on Growing Surfaces: A Molecular Dynamics Study.

We investigate the mechanism of a typical kinetic hydrate inhibitor (KHI), polyvinylcaprolactam (PVCap), which has been applied to prevent hydrate plugs from forming in gas pipe lines, using molecular dynamics simulations of crystal growth of ethylene oxide hydrate. Water-soluble ethylene oxide is chosen as a guest species to avoid problems associated with the presence of the gas phase in the simulation cell such as slow crystal growth. A PVCap dodecamer adsorbs irreversibly on the hydrate surface which grows at supercooling of 3 K when the hydrophobic part of two pendent groups are trapped in open cages at the surface. The amide hydrogen bonds make no contribution to the adsorption. PVCap can adsorb on various crystallographic planes of sI hydrate. This is in contrast to antifreeze proteins, each of which prefers a specific plane of ice. The trapped PVCap gives rise to necessarily the concave surface of the hydrate. The crystal growth rate decreases with increasing surface curvature, indicating that the inhibition by PVCap is explained by the Gibbs-Thomson effect.

String-like collective motion and diffusion in the interfacial region of ice.

We investigate collective molecular motion and the self-diffusion coefficient Ds of water molecules in the mobile interfacial layer of the secondary prismatic plane (112¯0) of hexagonal ice by molecular dynamics simulation based on the TIP4P/2005 water potential and a metrology of collective motion drawn from the field of glass-forming liquids. The width ξ of the mobile interfacial layer varies from a monolayer to a few nm as the temperature is increased towards the melting temperature Tm, in accordance with recent simulations and many experimental studies, although different experimental methods have differed in their precise estimates of the thickness of this layer. We also find that the dynamics within this mobile interfacial ice layer is "dynamically heterogeneous" in a fashion that has many features in common with glass-forming liquids and the interfacial dynamics of crystalline Ni over the same reduced temperature range, 2/3 < T/Tm < 1. In addition to exhibiting non-Gaussian diffusive transport, decoupling between mass diffusion and the structural relaxation time, and stretched exponential relaxation, we find string-like collective molecular exchange motion in the interfacial zone within the ice interfacial layer and colored noise fluctuations in the mean square molecular atomic displacement 〈u2〉 after a "caging time" of 1 ps, i.e., the Debye-Waller factor. However, while the heterogeneous dynamics of ice is clearly similar in many ways to molecular and colloidal glass-forming materials, we find distinct trends between the diffusion coefficient activation energy Ea for diffusion Ds and the interfacial width ξ from the scale of collective string-like motion L than those found in glass-forming liquids.

III 型不凍タンパク質の低濃度水溶液中で成長する 氷結晶ベーサル面上のピット形成過程の観察

III 型不凍タンパク質の低濃度水溶液中で成長する 氷結晶ベーサル面上のピット形成過程の観察

Interfacial Water Arrangement in the Ice-Bound State of an Antifreeze Protein: A Molecular Dynamics Simulation Study.

Molecular dynamics (MD) simulations have been carried out to study the heterogeneous ice nucleation on modeled peptide surfaces. Simulations show that large peptide surfaces made by TxT (threonine-x-threonine) motifs with the arrangements of threonine (Thr) residues identical to the periodic arrangements of waters on either the basal or prism plane of ice are capable of ice nucleation. Nucleated ice plane is the (0001) basal plane of hexagonal ice (Ih) or (111) plane of cubic ice (Ic). However, due to predefined simulation cell dimensions, the ice growth is only observed on the surface where the Thr residues are arranged like the water arrangement on the basal plane of ice Ih. The γ-methyl and γ-hydroxyl groups of Thr residue are necessary for such ice formation. From this ice nucleation and growth simulation, the interfacial water arrangement in the ice-bound state of Tenebrio molitor antifreeze protein (TmAFP) has been determined. The interfacial water arrangement in the ice-bound state of TmAFP is characterized by five-membered hydrogen bonded rings, where each of the hydroxyl groups of the Thr residues on the ice-binding surface (IBS) of the protein is a ring member. It is found that the water arrangement at the protein-ice interface is distorted from that in bulk ice. Our analysis further reveals that the hydroxyl groups of Thr residues on the IBS of TmAFP form maximum three hydrogen bonds each with the waters in the bound state and methyl groups of Thr residues occupy wider spaces than the normal grooves on the (111) plane of ice Ic. Methyl groups are also located above and along the 3-fold rotational axes of the chair-formed hexagonal hydrogen bonded water rings on the (111) plane.

Ice Nucleation Efficiency of Hydroxylated Organic Surfaces Is Controlled by Their Structural Fluctuations and Mismatch to Ice.

Heterogeneous nucleation of ice induced by organic materials is of fundamental importance for climate, biology, and industry. Among organic ice-nucleating surfaces, monolayers of long chain alcohols are particularly effective, while monolayers of fatty acids are significantly less so. As these monolayers expose to water hydroxyl groups with an order that resembles the one in the basal plane of ice, it was proposed that lattice matching between ice and the surface controls their ice-nucleating efficiency. Organic monolayers are soft materials and display significant fluctuations. It has been conjectured that these fluctuations assist in the nucleation of ice. Here we use molecular dynamic simulations and laboratory experiments to investigate the relationship between the structure and fluctuations of hydroxylated organic surfaces and the temperature at which they nucleate ice. We find that these surfaces order interfacial water to form domains with ice-like order that are the birthplace of ice. Both mismatch and fluctuations decrease the size of the preordered domains and monotonously decrease the ice freezing temperature. The simulations indicate that fluctuations depress the freezing efficiency of monolayers of alcohols or acids to half the value predicted from lattice mismatch alone. The model captures the experimental trend in freezing efficiencies as a function of chain length and predicts that alcohols have higher freezing efficiency than acids of the same chain length. These trends are mostly controlled by the modulation of the structural mismatch to ice. We use classical nucleation theory to show that the freezing efficiencies of the monolayers are directly related to their free energy of binding to ice. This study provides a general framework to relate the equilibrium thermodynamics of ice binding to a surface and the nonequilibrium ice freezing temperature and suggests that these could be predicted from the structure of interfacial water.

Concentration-dependent oligomerization of an alpha-helical antifreeze polypeptide makes it hyperactive

A supersoluble 40-residue type I antifreeze protein (AFP) was discovered in a righteye flounder, the barfin plaice (bp). Unlike all other AFPs characterized to date, bpAFP transitions from moderately-active to hyperactive with increasing concentration. At sub-mM concentrations, bpAFP bound to pyramidal planes of ice to shape it into a bi-pyramidal hexagonal trapezohedron, similarly to the other moderately-active AFPs. At mM concentrations, bpAFP uniquely underwent further binding to the whole ice crystal surface including the basal planes. The latter caused a bursting ice crystal growth normal to c-axis, 3 °C of high thermal hysteresis, and alteration of an ice crystal into a smaller lemon-shaped morphology, all of which are well-known properties of hyperactive AFPs. Analytical ultracentrifugation showed this activity transition is associated with oligomerization to form tetramer, which might be the forerunner of a naturally occurring four-helix-bundle AFP in other flounders.