Hyperactive Antifreeze Proteins Research Articles

Extended Temperature Range of the Ice-Binding Protein Activity.

Ice-binding proteins (IBPs) are expressed in various organisms for several functions, such as protecting them from freezing and freeze injuries. Via adsorption on ice surfaces, IBPs depress ice growth and recrystallization and affect nucleation and ice shaping. IBPs have shown promise in mitigating ice growth under moderate supercooling conditions, but their functionality under cryogenic conditions has been less explored. In this study, we investigate the impact of two types of antifreeze proteins (AFPs): type III AFP from fish and a hyperactive AFP from an insect, the Tenebrio molitor AFP, in vitrified dimethylsulfoxide (DMSO) solutions. We report that these AFPs depress devitrification at -80 °C. Furthermore, in cases where devitrification does occur, AFPs depress ice recrystallization during the warming stage. The data directly demonstrate that AFPs are active at temperatures below the regime of homogeneous nucleation. This research paves the way for exploring AFPs as potential enhancers of cryopreservation techniques, minimizing ice-growth-related damage, and promoting advancements in this vital field.

Physics of Ice Nucleation and Antinucleation: Action of Ice-Binding Proteins.

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

Synergistic Effect of Hyperactive Antifreeze Protein on Inhibition of Gas-Hydrate Growth by Hydrophobic and Hydrophilic Groups.

Antifreeze proteins (AFPs) are biodegradable inhibitors that effectively prevent the formation of natural gas hydrates that block pipelines. In this study, molecular dynamics simulations were employed to establish a kinetic model of the hyperactive insect antifreeze protein (Tenebrio molitor, TmAFP) and its mutants to inhibit the growth of sI natural methane hydrate. Simulations revealed that the hydrophobic and hydrophilic groups of threonine (Thr) residues at hydrate-binding sites played a synergistic role in binding hydrates. The hydrophobic groups anchored TmAFP to the hydrate surface through residues Thr39-Thr65 by migrating pendant hydrophobic methyl groups to the hydrate semicages. The hydrophilic groups stabilized TmAFP by hydrogen bonding with water molecules and integrating them into a quasi-hydrate structure, which more effectively inhibited hydrate growth. The results suggest that the hydrate growth inhibition is attributed to both the shape complementarity and the flexibility of binding residues. The synergy between hydrophobic and hydrophilic groups provides guidance for the design of more effective hydrate inhibitors.

Chilling injury in human kidney tubule cells after subzero storage is not mitigated by antifreeze protein addition

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temperatures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SZ-SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to static cold storage (SCS). Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of SZ-SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 °C) and at −6 °C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 °C, those stored at −6 °C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable SZ-SCS may not be effective for increasing storage times.

Elucidating the Sluggish Water Dynamics at the Ice-Binding Surface of the Hyperactive Tenebrio molitor Antifreeze Protein.

Quasi-ice-like hydration waters on the ice-binding surface (IBS) of an antifreeze protein (AFP) commonly exhibit sluggish dynamics especially at low temperatures. In this work, we have analyzed molecular dynamics (MD) simulation trajectories at two different temperatures for Tenebrio molitor antifreeze protein (TmAFP) to explore whether the unique quasi-ice-like structuring of hydration water has any impact on making their dynamics slower on the IBS of the protein. Our calculation reveals that, as translational dynamics is coupled with the conformational fluctuations, hydration water on the IBS exhibits sluggish translational motion due to reduced flexibility of the IBS compared to that on the non-ice-binding surface (NIBS) of the protein. Interestingly, it is noticed that rotational motion of hydration water is not coupled with the conformational fluctuations of the surfaces. In that case, structural relaxations of the protein-water (PW) and water-water (WW) hydrogen bonds compete with each other to make the rotational dynamics of hydration water around the IBS either faster or slower with respect to those around the NIBS. At low temperature, the slower structural relaxation of water-water hydrogen bonds dominates and imparts sluggish rotational motion of the hydration water on the IBS of the protein. The slower structural relaxation of water-water hydrogen bonds and hence the retarded rotational dynamics, despite the weak short-lived PW hydrogen bonds on the IBS, is clearly a manifestation of the rigid quasi-ice-like structure of the hydration shell on that surface.

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.

Residue-specific structure and dynamics of water around the insect antifreeze protein, DAFP-1: a molecular dynamics study

Antifreeze proteins are a class of proteins across various species that exhibit thermal hysteresis. The mechanisms by which these proteins prevent freezing without altering the melting point has yet to be fully characterized and understood. DAFP-1 is a hyperactive antifreeze protein present in the beetle, Dendroides canadensis and is modelled using the Amber99 force field. We present residue specific methods of analysis to isolate amino acids in the hypothesized ice binding face and the non binding face.

Do antifreeze proteins generally possess the potential to promote ice growth?

The binding of antifreeze proteins (AFPs) to ice needs to be mediated by interfacial water molecules. Our previous study of the effect of AFPs on the dynamics of the interfacial water of freezing at its initial stage has shown that AFPs can promote the growth of ice before binding to it. However, whether different AFPs can promote the freezing of water molecules on the basal and the prismatic surfaces of ice still needs further study. In the present contribution, five representative natural AFPs with different structures and different activities that can be adsorbed on the basal and/or prismatic surfaces of ice are investigated at the atomic level. Our results show that the phenomenon of promoting the growth of ice crystals is not universal. Only hyperactive AFPs (hypAFPs) can promote the growth of the basal plane of ice, while moderately active AFPs cannot. Moreover, this significant promotion is not observed on the prismatic plane regardless of their activity. Further analysis indicates that this promotion may result from the thicker ice/water interface of the basal plane, and the synergy of hypAFPs with ice crystals.

Hyperactive Antifreeze Proteins Promote Ice Growth before Binding to It.

The antifreeze mechanism of antifreeze proteins (AFPs) evolved by organisms has been widely studied. However, detailed knowledge of the synergy between AFPs and ice crystals still remains fragmentary. In the present contribution, the cooperative effect of the hyperactive insect antifreeze protein TmAFP and ice crystals on the interfacial water during the entire process of inhibiting ice growth is systematically investigated at the atomic level and compared with its low activity mutant and a nonantifreeze protein. The results indicate a significant synergy between TmAFP and ice crystals, which enables the TmAFP to promote the ice growth before adsorbing on the surfaces of the ice crystals, while the mutant and the nonantifreeze protein cannot promote the ice growth due to the lack of this synergy. When TmAFP approaches the ice surface, the interfacial water is induced by both the AFP and the ice crystals to form the anchored clathrate motif, which binds TmAFP to the ice surface, resulting in a local increase in the curvature of the ice surface, thereby inhibiting the growth of ice. In this study, three stages, namely, promotion, adsorption, and inhibition, are observed in the complete process of TmAFP inhibiting ice growth, and the synergistic mechanism between protein and ice crystals is revealed. The results are helpful for the design of antifreeze proteins and bioinspired antifreeze materials with superior performance.

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

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

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

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

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated −5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6−8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo­ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

Deciphering the Role of the Non-ice-binding Surface in the Antifreeze Activity of Hyperactive Antifreeze Proteins.

Antifreeze proteins (AFPs) show thermal hysteresis through specific interaction with the ice crystal. Hyperactive AFPs interact with the ice surface through a threonine-rich motif present at their ice-binding surface (IBS). Ordering of water around the IBS was extensively investigated. However, the role of non-IBS in ice growth inhibition is yet to be understood completely. The present study explores the nature of hydration and its length-scale evaluation around the non-IBS for hyperactive AFPs. We observed that the hydration layer of non-IBS is liquid-like, even in highly supercooled conditions, and the nature of hydration is drastically different from the hydration pattern of non-AFP surfaces. In similar conditions, the hydration layer around the IBS is ice-like ordered. Non-IBS of the hyperactive AFP exposes toward the bulk and is able to maintain the liquid-like character of its hydration water up to 15 Å. We also find that the amino acid compositions and their spatial distribution on the non-IBS are markedly different from those of the IBS and non-AFP surfaces. These results elucidate the combined role of IBS and non-IBS in ice-growth inhibition. While IBS is required to adsorb on ice efficiently, the exposed non-IBS may prevent ice nucleation/growth on top of the bound AFPs.

Interfacial water controls the process of adsorption of hyperactive antifreeze proteins onto the ice surface

A mechanism of interactions between the ice-binding surface of a hyperactive antifreeze protein molecule and the ice surface is proposed, involving the influence of water present between the two surfaces on the behavior of the approaching molecule. It is demonstrated that the interfacial water, even before its full solidification, can act as a factor that pushes away or pulls nearer the protein molecule to ensure its proper positioning. It is possible thanks to the structural properties of interfacial water. These properties include the ability to create high-volume aggregates of water molecules. They can appear near and be anchored to both the ice-binding plane of the antifreeze molecule and the ice surface. When an AFP approaches the growing face of ice, these high-volume, ordered structures near the ice and near the AFP molecule merge together smoothly, but only if the proper distance between the ice and the AFP is ensured. If this is not the case, the resulting merged structure is deformed from its preferred shape and as a result a force occurs that attempts to correct the positioning of the protein. Only then the crystallization of the merged aggregate can proceed unhampered which results in binding of the AFP molecule onto the ice. • The molecular mechanism of interaction of antifreeze proteins with the surface of ice remains unclear. • The existence of long-ranged, water-mediated interactions between the AFP molecule and the ice surface is demonstrated. • These interactions affect the positioning of the AFP molecule relatively to the ice and influence the adsorption process. • Observed behavior is explained by evoking specific structural properties of liquid water.

Role of the Solvation Water in Remote Interactions of Hyperactive Antifreeze Proteins with the Surface of Ice.

Most protein molecules do not adsorb onto ice, one of the exceptions being so-called antifreeze proteins. In this paper, we describe that there is a force pushing an antifreeze protein molecule away from the ice surface when it is not oriented with its ice-binding plane toward the ice and that this pushing force may be also present even when the protein is oriented with its ice-binding plane toward the ice. This force is absent only when certain specific distance criteria are met, regarding the surface of ice and the protein. It acts at early stages of adsorption, prior to the solidification of water between the ice and the protein molecule nearby. We propose the water-originating mechanism of the generation of this force and also the mechanism of remote attachment of an antifreeze molecule to the ice surface. In liquid water, there exist locally favored structures, ordered and of high specific volume. The presence of a protein molecule usually shifts the equilibrium that exists in liquid water toward increasing the number of high-density, disordered structures and diminishing the number of low-density structures. Creation of the locally favored structures may be hampered not only near the non-ice-binding surfaces but also between the ice surface and the protein surface, if the distance between these surfaces does not allow these structures to develop because the available space is not sufficient for their proper formation. This conclusion is supported by the analysis of the mean geometry of a single hydrogen bond, as well as of the hydrogen bond network in the solvation layer and a structural order parameter that characterizes the separation between the first and second solvation shells of a water molecule.

Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology.

Micromolar concentrations of hyperactive antifreeze proteins (AFPs) from insects can prevent aqueous solutions from freezing down to at least −6 °C. To explore cryopreservation of cells, tissues and organs at these temperatures without ice formation, we have developed a protocol to reliably produce ultrapure Tenebrio molitor AFP from cold-acclimated beetle larvae reared in the laboratory. The AFP was prepared from crude larval homogenates through five cycles of rotary ice-affinity purification, which can be completed in one day. Recovery of the AFP at each step was >90% and no impurities were detected in the final product. The AFP is a mixture of isoforms that are more active in combination than any one single component. Toxicity testing of the purified AFP in cell culture showed no inhibition of cell growth. The production process can easily be scaled up to industrial levels, and the AFP used in cryobiology applications was recovered for reuse in good yield and with full activity.

Hydrogen-Bonding and Hydrophobic Groups Contribute Equally to the Binding of Hyperactive Antifreeze and Ice-Nucleating Proteins to Ice.

Hyperactive insect antifreeze proteins and bacterial ice-nucleating proteins are arguably the most potent ice-binding molecules in nature. These highly effective proteins bind ice through amphiphilic ice-binding sites based on arrays of threonine residues. It remains poorly understood how hydrophilic and hydrophobic groups of the binding site contribute to the ice affinity of proteins. Here, we use molecular simulations to demonstrate that the hydrogen-bonding and hydrophobic groups at the ice-binding site of the antifreeze protein TmAFP of Tenebrio molitor and extended ice-nucleating protein surfaces contribute distinctively yet almost equally in magnitude to their binding free energy to ice. The methyl groups rigidize the ice-binding site, slow the water dynamics at the ice-binding surface, and stabilize the clathrate-like water in the anchored clathrate motif that binds these proteins to ice. We find that hydrophobic dehydration of the methyl group does not contribute to the binding free energy of the protein to ice. The role of the hydroxyl groups is to anchor the clathrate-like water through direct hydrogen-bonding, positioning and slowing the dynamics of water at the trough of the binding site. We uncover a correlation between slower dynamics of water at the binding site for the protein in solution and stronger free energy of binding of the protein to ice. The synergy between hydrophobic and hydrophilic groups unveiled by this study provides guidance for the design of synthetic ice-binding molecules with enhanced ice nucleation and antifreeze activity.

How Size and Aggregation of Ice-Binding Proteins Control Their Ice Nucleation Efficiency.

Organisms that thrive at cold temperatures produce ice-binding proteins to manage the nucleation and growth of ice. Bacterial ice-nucleating proteins (INP) are typically large and form aggregates in the cell membrane, while insect hyperactive antifreeze proteins (AFP) are soluble and generally small. Experiments indicate that larger ice-binding proteins and their aggregates nucleate ice at warmer temperatures. Nevertheless, a quantitative understanding of how size and aggregation of ice-binding proteins determine the temperature Thet at which proteins nucleate ice is still lacking. Here, we address this question using molecular simulations and nucleation theory. The simulations indicate that the 2.5 nm long antifreeze protein TmAFP nucleates ice at 2 ± 1 °C above the homogeneous nucleation temperature, in good agreement with recent experiments. We predict that the addition of ice-binding loops to TmAFP increases Thet, but not enough to compete in efficiency with the bacterial INP. We implement an accurate procedure to determine Thet of surfaces of finite size using classical nucleation theory, and, after validating the theory against Thet of the proteins in molecular simulations, we use it to predict Thet of the INP of Ps. syringae as a function of the length and number of proteins in the aggregates. We conclude that assemblies with at most 34 INP already reach the Thet = -2 °C characteristic of this bacterium. Interestingly, we find that Thet is a strongly varying nonmonotonic function of the distance between proteins in the aggregates. This indicates that, to achieve maximum freezing efficiency, bacteria must exert exquisite, subangstrom control of the distance between INP in their membrane.

Molecular structure of a hyperactive antifreeze protein adsorbed to ice.

Antifreeze proteins (AFPs) are a unique class of proteins that bind to ice crystal surfaces and arrest their growth. The working mechanism of AFPs is not well understood because, as of yet, it was not possible to perform molecular-scale studies of AFPs adsorbed to the surface of ice. Here, we study the structural properties of an AFP from the insect Rhagium mordax (RmAFP) adsorbed to ice with surface specific heterodyne-detected vibrational sum-frequency generation spectroscopy and molecular dynamic simulations. We find that RmAFP, unlike other proteins, retains its hydrating water molecules upon adsorption to the ice surface. This hydration water has an orientation and hydrogen-bond structure different from the ice surface, thereby inhibiting the insertion of water layers in between the protein and the ice surface.

Collective Transformation of Water between Hyperactive Antifreeze Proteins: RiAFPs

We demonstrate, by molecular dynamics simulations, that water confined between a pair of insect hyperactive antifreeze proteins from the longhorn beetle Rhagium inquisitor is discontinuously expelled as the two proteins approach each other at a certain distance. The extensive striped hydrophobic–hydrophilic pattern on the surface, comprising arrays of threonine residues, enables water to form three independent ice channels through the assistance of hydroxyl groups, even at 300 K. The transformation is reminiscent of a freezing–melting transition rather than a drying transition and governs the stable protein–protein separation in the evaluation of the potential of mean force. The collectivity of water penetration or expulsion and the hysteresis in the time scale of ten nanoseconds predict a potential first-order phase transition at the limit of infinite size and provide a new framework for the water-mediated interaction between solutes.