Ice-Binding Proteins and Their Function.

Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.

On the engulfment of antifreeze proteins by ice.

Antifreeze proteins (AFPs) are remarkable biomolecules that suppress ice formation at trace concentrations. To inhibit ice growth, AFPs must not only bind to ice crystals, but also resist engulfment by ice. The highest supercooling, [Formula: see text], for which AFPs are able to resist engulfment is widely believed to scale as the inverse of the separation, [Formula: see text], between bound AFPs, whereas its dependence on the molecular characteristics of the AFP remains poorly understood. By using specialized molecular simulations and interfacial thermodynamics, here, we show that in contrast with conventional wisdom, [Formula: see text] scales as [Formula: see text] and not as [Formula: see text]. We further show that [Formula: see text] is proportional to AFP size and that diverse naturally occurring AFPs are optimal at resisting engulfment by ice. By facilitating the development of AFP structure-function relationships, we hope that our findings will pave the way for the rational design of AFPs.

Nanoscopy of single antifreeze proteins reveals that reversible ice binding is sufficient for ice recrystallization inhibition but not thermal hysteresis.

Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.

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.

Diverse Origins of Near-Identical Antifreeze Proteins in Unrelated Fish Lineages Provide Insights Into Evolutionary Mechanisms of New Gene Birth and Protein Sequence Convergence.

Determining the origins of novel genes and the mechanisms driving the emergence of new functions is challenging yet crucial for understanding evolutionary innovations. Recently evolved fish antifreeze proteins (AFPs) offer a unique opportunity to explore these processes, particularly the near-identical type I AFP (AFPI) found in four phylogenetically divergent fish taxa. This study tested the hypothesis of protein sequence convergence beyond functional convergence in three unrelated AFPI-bearing fish lineages. Through comprehensive comparative analyses of newly sequenced genomes of winter flounder and grubby sculpin, along with available high-quality genomes of cunner and 14 other related species, the study revealed that near-identical AFPI proteins originated from distinct genetic precursors in each lineage. Each lineage independently evolved a de novo coding region for the novel ice-binding protein while repurposing fragments from their respective ancestors into potential regulatory regions, representing partial de novo origination-a process that bridges de novo gene formation and the neofunctionalization of duplicated genes. The study supports existing models of new gene origination and introduces new ones: the innovation-amplification-divergence model, where novel changes precede gene duplication; the newly proposed duplication-degeneration-divergence model, which describes new functions arising from degenerated pseudogenes; and the duplication-degeneration-divergence gene fission model, where each new sibling gene differentially degenerates and renovates distinct functional domains from their parental gene. These findings highlight the diverse evolutionary pathways through which a novel functional gene with convergent sequences at the protein level can evolve across divergent species, advancing our understanding of the mechanistic intricacies in new gene formation.

De novo assembly and annotation of the Patagonian toothfish (Dissostichus eleginoides) genome.

BACKGROUND: Patagonian toothfish (Dissostichus eleginoides) is an economically and ecologically important fish species in the family Nototheniidae. Juveniles occupy progressively deeper waters as they mature and grow, and adults have been caught as deep as 2500 m, living on or in just above the southern shelves and slopes around the sub-Antarctic islands of the Southern Ocean. As apex predators, they are a key part of the food web, feeding on a variety of prey, including krill, squid, and other fish. Despite its importance, genomic sequence data, which could be used for more accurate dating of the divergence between Patagonian and Antarctic toothfish, or establish whether it shares adaptations to temperature with fish living in more polar or equatorial climes, has so far been limited. RESULTS: A high-quality D. eleginoides genome was generated using a combination of Illumina, PacBio and Omni-C sequencing technologies. To aid the genome annotation, the transcriptome derived from a variety of toothfish tissues was also generated using both short and long read sequencing methods. The final genome assembly was 797.8 Mb with a N50 scaffold length of 3.5 Mb. Approximately 31.7% of the genome consisted of repetitive elements. A total of 35,543 putative protein-coding regions were identified, of which 50% have been functionally annotated. Transcriptomics analysis showed that approximately 64% of the predicted genes (22,617 genes) were found to be expressed in the tissues sampled. Comparative genomics analysis revealed that the anti-freeze glycoprotein (AFGP) locus of D. eleginoides does not contain any AFGP proteins compared to the same locus in the Antarctic toothfish (Dissostichus mawsoni). This is in agreement with previously published results looking at hybridization signals and confirms that Patagonian toothfish do not possess AFGP coding sequences in their genome. CONCLUSIONS: We have assembled and annotated the Patagonian toothfish genome, which will provide a valuable genetic resource for ecological and evolutionary studies on this and other closely related species.

Design and Mechanistic Insights into α-Helical p-Terphenyl Guanidines as Potent Small-Molecule Antifreeze Agents.

Ice formation is a critical challenge across multiple fields, from industrial applications to biological preservation. Inspired by natural antifreeze proteins, we designed and synthesized a new class of small-molecule antifreezes based on α-helical p-terphenyl scaffolds with guanidine side chains. These p-terphenyl guanidines 1, among the smallest molecules that mimic α-helical structures, exhibit potent ice recrystallization inhibition (IRI) activity, similar to that of existing large α-helical antifreeze compounds. The most effective compound, 1a, with four C1-carbon guanidine moieties, demonstrated a superior IRI activity of 0.46 (1 mg/mL). Using molecular dynamics simulations with density-functional theory and separate pKa calculations, we elucidated the mechanisms underlying their antifreeze properties.

Genomics of Secondarily Temperate Adaptation in the Only Non-Antarctic Icefish.

White-blooded Antarctic icefishes, a family within the adaptive radiation of Antarctic notothenioid fishes, are an example of extreme biological specialization to both the chronic cold of the Southern Ocean and life without hemoglobin. As a result, icefishes display derived physiology that limits them to the cold and highly oxygenated Antarctic waters. Against these constraints, remarkably one species, the pike icefish Champsocephalus esox, successfully colonized temperate South American waters. To study the genetic mechanisms underlying secondarily temperate adaptation in icefishes, we generated chromosome-level genome assemblies of both C. esox and its Antarctic sister species, Champsocephalus gunnari. The C. esox genome is similar in structure and organization to that of its Antarctic congener; however, we observe evidence of chromosomal rearrangements coinciding with regions of elevated genetic divergence in pike icefish populations. We also find several key biological pathways under selection, including genes related to mitochondria and vision, highlighting candidates behind temperate adaptation in C. esox. Substantial antifreeze glycoprotein (AFGP) pseudogenization has occurred in the pike icefish, likely due to relaxed selection following ancestral escape from Antarctica. The canonical AFGP locus organization is conserved in C. esox and C. gunnari, but both show a translocation of two AFGP copies to a separate locus, previously unobserved in cryonotothenioids. Altogether, the study of this secondarily temperate species provides an insight into the mechanisms underlying adaptation to ecologically disparate environments in this otherwise highly specialized group.

Horizontal Gene Transfer in Vertebrates: A Fishy Tale.

The recent assembly of the herring genome suggests this fish acquired its antifreeze protein gene by horizontal transfer and then passed a copy on to the smelt. The direction of gene transfer is confirmed by some accompanying transposable elements and by the breakage of gene synteny.

Engineered Compounds to Control Ice Nucleation and Recrystallization.

One of the greatest concerns in the subzero storage of cells, tissues, and organs is the ability to control the nucleation or recrystallization of ice. In nature, evidence of these processes, which aid in sustaining internal temperatures below the physiologic freezing point for extended periods of time, is apparent in freeze-avoidant and freeze-tolerant organisms. After decades of studying these proteins, we now have easily accessible compounds and materials capable of recapitulating the mechanisms seen in nature for biopreser-vation applications. The output from this burgeoning area of research can interact synergistically with other novel developments in the field of cryobiology, making it an opportune time for a review on this topic.

From freezing to functioning: cellular strategies of cold-adapted bacteria for surviving in extreme environments.

The ability of cold-adapted bacteria to survive in extreme cold and diverse temperatures is due to their unique attributes like cell membrane stability, up-regulation of peptidoglycan biosynthesis, increased production of extracellular polymeric substances, and expansion of membrane pigment. Various cold-adapted proteins, including ice-nucleating proteins (INPs), antifreeze proteins (AFPs), cold shock proteins (Csps), and cold-acclimated proteins (CAPs), help the bacteria to survive in these environments. To sustain cells from extreme cold conditions and maintain stability in temperature fluctuations, survival strategies at the molecular level and their mechanism play significant roles in adaptations in cryospheric conditions. Furthermore, cold shock domains present in the multifunctional cold shock proteins play crucial roles in their adaptation strategies. The considerable contribution of lipopeptides, osmolytes, and membrane pigments plays an integral part in their survival in extreme environments. This review summarizes the evolutionary history of cold-adapted bacteria and their molecular and cellular adaptation strategies to thrive in harsh cold environments. It also discusses the importance of carotenoids produced, lipid composition, cryoprotectants, proteins, and chaperones related to this adaptation. Furthermore, the functions and mechanisms of adaptations within the cell are discussed briefly. One can utilize and explore their potential in various biotechnology applications and their evolutionary journey by knowing the inherent mechanism of their molecular and cellular adaptation to cold climatic conditions. This review will help all branches of the life science community understand the basic microbiology of psychrophiles and their hidden prospect in life science research.

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.

Protein inclusion into ice can dissociate subunits.

An antifreeze protein's inclusion into ice can be used to purify it from other proteins and solutes. Domains that are covalently attached to the antifreeze protein are also drawn into the ice such that the ice-binding portion of the fusion protein can be used as an affinity tag. Here we have explored the use of ice-affinity tags on multi-subunit proteins. When an ice-binding protein was attached as a tag to multisubunit complexes a substantial portion of each multimer dissociated during overgrowth by the ice. The protein subunit attached to the affinity tag was enriched in the ice and the other subunit was appreciably excluded. We suggest that step growth of the advancing ice front generates shearing forces on the bound complex that can disrupt non-covalent protein-protein interactions. This will effectively limit the use of ice-affinity tags to single subunit proteins.

Glycosylated Polyhydroxyproline Is a Potent Antifreeze Molecule.

Molecules that inhibit the growth of ice crystals are highly desirable for applications in building materials, foods, and agriculture. Antifreezes are particularly essential in biomedicine for tissue banking, yet molecules currently in use have known toxic effects. Antifreeze glycoproteins have evolved naturally in polar fish species living in subzero climates, but practical issues with collection and purification have limited their commercial use. Here, we present a synthetic strategy using polymerization of amino acid N-carboxyanhydrides to produce polypeptide mimics of these potent natural antifreeze proteins. We investigated a set of mimics with varied structural properties and identified a glycopolypeptide with potent ice recrystallization inhibition properties. We optimized for molecular weight, characterized their conformations, and verified their cytocompatibility in a human cell line. Overall, we present a material that will have broad applications as a biocompatible antifreeze.

Antifreeze Polysaccharides from Wheat Bran: The Structural Characterization and Antifreeze Mechanism.

Exploring a novel natural cryoprotectant and understanding its antifreeze mechanism allows the rational design of future sustainable antifreeze analogues. In this study, various antifreeze polysaccharides were isolated from wheat bran, and the antifreeze activity was comparatively studied in relation to the molecular structure. The antifreeze mechanism was further revealed based on the interactions of polysaccharides and water molecules through dynamic simulation analysis. The antifreeze polysaccharides showed distinct ice recrystallization inhibition activity, and structural analysis suggested that the polysaccharides were arabinoxylan, featuring a xylan backbone with a majority of Araf and minor fractions of Manp, Galp, and Glcp involved in the side chain. The antifreeze arabinoxylan, characterized by lower molecular weight, less branching, and more flexible conformation, could weaken the hydrogen bonding of the surrounding water molecules more evidently, thus retarding the transformation of water molecules into the ordered ice structure.

Synergistic Effects of the Superhydrophilic and Superhydrophobic Components on the Antifreezing Performances of Latex Particles and Anti-Icing Properties of Latex Films.

The development of new materials for antifreezing and anti-icing applications is a big challenge in industry and academic area. Inspired by the antifreeze proteins, latex particles with superhydrophilic zwitterionic shells and superhydrophobic cores are synthesized by reversible addition-fragmentation chain transfer emulsion polymerization, and the applications of the latex particles in antifreezing and anti-icing applications are investigated. In antifreezing study, the critical aggregate temperature (CAT) of the latex particles decreases, and the separation of the melting and freezing temperature of ice increases with the particle concentration. Enzyme molecules can be cryopreserved in the particle solution, and their bioactivities are well maintained. Latex particles are casted into latex films with dynamic surfaces. Anti-icing performances, including antifrosting properties, freezing delay time, and ice adhesion strengths, are studied; and the water-treated latex films present stronger anti-icing properties than other films, due to the synergistic effects of the superhydrophilic and superhydrophobic components. In addition, latex particles with zwitterionic shells and poly(n-butyl methacrylate) cores, and latex particles with small molecular surfactant on the surfaces are synthesized. The antifreezing performances of the latex particles and anti-icing properties of the latex films are compared.

Frost fighters: unveiling the potential of microbial antifreeze proteins in biotech innovation.

Polar environments pose extreme challenges for life due to low temperatures, limited water, high radiation, and frozen landscapes. Despite these harsh conditions, numerous macro and microorganisms have developed adaptive strategies to reduce the detrimental effects of extreme cold. A primary survival tactic involves avoiding or tolerating intra and extracellular freezing. Many organisms achieve this by maintaining a supercooled state by producing small organic compounds like sugars, glycerol, and amino acids, or through increasing solute concentration. Another approach is the synthesis of ice-binding proteins, specifically antifreeze proteins (AFPs), which hinder ice crystal growth below the melting point. This adaptation is crucial for preventing intracellular ice formation, which could be lethal, and ensuring the presence of liquid water around cells. AFPs have independently evolved in different species, exhibiting distinct thermal hysteresis and ice structuring properties. Beyond their ecological role, AFPs have garnered significant attention in biotechnology for potential applications in the food, agriculture, and pharmaceutical industries. This review aims to offer a thorough insight into the activity and impacts of AFPs on water, examining their significance in cold-adapted organisms, and exploring the diversity of microbial AFPs. Using a meta-analysis from cultivation-based and cultivation-independent data, we evaluate the correlation between AFP-producing microorganisms and cold environments. We also explore small and large-scale biotechnological applications of AFPs, providing a perspective for future research.

Long-range, water-mediated interaction between a moderately active antifreeze protein molecule and the surface of ice.

Using molecular dynamics simulations, we show that a molecule of moderately active antifreeze protein (type III AFP, QAE HPLC-12 isoform) is able to interact with ice in an indirect manner. This interaction occurs between the ice binding site (IBS) of the AFP III molecule and the surface of ice, and it is mediated by liquid water, which separates these surfaces. As a result, the AFP III molecule positions itself at a specific orientation and distance relative to the surface of ice, which enables the effective binding (via hydrogen bonds) of the molecule with the nascent ice surface. Our results show that the final adsorption of the AFP III molecule on the surface of ice is not achieved by chaotic diffusion movements, but it is preceded by a remote, water-mediated interaction between the IBS and the surface of ice. The key factor that determines the existence of this interaction is the ability of water molecules to spontaneously form large, high-volume aggregates that can be anchored to both the IBS of the AFP molecule and the surface of ice. The results presented in this work for AFP III are in full agreement with the ones obtained by us previously for hyperactive CfAFP, which indicates that the mechanism of the remote interaction of these molecules with ice remains unchanged despite significant differences in the molecular structure of their ice binding sites. For that reason, we can expect that also other types of AFPs interact with the ice surface according to an analogous mechanism.

Solvation of molecules from the family of "domain of unknown function" 3494 and their ability to bind to ice.

In 2012, the molecular structure of a new, broad class of ice-binding proteins, classified as "domain of unknown function" (DUF) 3494, was described for the first time. These proteins have a common tertiary structure and are characterized by a very wide spectrum of antifreeze activity (from weakly active to hyperactive). The ice-binding surface (IBS) region of these molecules differs significantly in its structure from the IBS of previously known antifreeze proteins (AFPs), showing a complete lack of regularity and high hydrophilicity. The presence of a regular, repeating structural motif in the IBS region of hitherto known AFP molecules, combined with the hydrophobic nature of this surface, promotes the formation of an ice-like ordering of the solvation water layer and, as a result, facilitates the process of transformation of this water layer into ice. It is, therefore, surprising that the newly discovered DUF3494 class of proteins clearly breaks out of this characteristic. In this paper, using molecular dynamics simulations, we analyze the solvation water structure of the IBS region of both DUF3494 family molecules and AFPs. As we show, although the IBS of DUF3494 molecules does not form an ice-like water structure in the solvation layer, this is compensated by the formation of the equivalent of "anchored clathrate water," in the form of a relatively large number of water molecules bound to the surface of the protein molecule and providing potential binding sites for it to the ice surface.

Design of eco-friendly antifreeze peptides as novel inhibitors of gas-hydration kinetics.

In this study, peptides designed using fragments of an antifreeze protein (AFP) from the freeze-tolerant insect Tenebrio molitor, TmAFP, were evaluated as inhibitors of clathrate hydrate formation. It was found that these peptides exhibit inhibitory effects by both direct and indirect mechanisms. The direct mechanism involves the displacement of methane molecules by hydrophobic methyl groups from threonine residues, preventing their diffusion to the hydrate surface. The indirect mechanism is characterized by the formation of cylindrical gas bubbles, the morphology of which reduces the pressure difference at the bubble interface, thereby slowing methane transport. The transfer of methane to the hydrate interface is primarily dominated by gas bubbles in the presence of antifreeze peptides. Spherical bubbles facilitate methane migration and potentially accelerate hydrate formation; conversely, the promotion of a cylindrical bubble morphology by two of the designed systems was found to mitigate this effect, leading to slower methane transport and reduced hydrate growth. These findings provide valuable guidance for the design of effective peptide-based inhibitors of natural-gas hydrate formation with potential applications in the energy and environmental sectors.