Functional aggregation of cell-free proteins enables fungal ice nucleation.

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits. Fusarium INs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs.

Ice Recrystallization Inhibition Is Insufficient to Explain Cryopreservation Abilities of Antifreeze Proteins.

Antifreeze proteins (AFPs) and glycoproteins (AFGPs) are exemplary at modifying ice crystal growth and at inhibiting ice recrystallization (IRI) in frozen solutions. These properties make them highly attractive for cold storage and cryopreservation applications of biological tissue, food, and other water-based materials. The specific requirements for optimal cryostorage remain unknown, but high IRI activity has been proposed to be crucial. Here, we show that high IRI activity alone is insufficient to explain the beneficial effects of AF(G)Ps on human red blood cell (hRBC) survival. We show that AF(G)Ps with different IRI activities cause similar cell recoveries of hRBCs and that a modified AFGP variant with decreased IRI activity shows increased cell recovery. The AFGP variant was found to have enhanced interactions with a hRBC model membrane, indicating that the capability to stabilize cell membranes is another important factor for increasing the survival of cells after cryostorage. This information should be considered when designing novel synthetic cryoprotectants.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP's unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.

Inhibition of Bacterial Ice Nucleators Is Not an Intrinsic Property of Antifreeze Proteins.

Cold-adapted organisms use antifreeze proteins (AFPs) or ice-nucleating proteins (INPs) for the survival in freezing habitats. AFPs have been reported to be able to inhibit the activity of INPs, a property that would be of great physiological relevance. The generality of this effect is not understood, and for the few known examples of INP inhibition by AFPs, the molecular mechanisms remain unclear. Here, we report a comprehensive evaluation of the effects of five different AFPs on the activity of bacterial ice nucleators using a high-throughput ice nucleation assay. We find that bacterial INPs are inhibited by certain AFPs, while others show no effect. Thus, the ability to inhibit the activity of INPs is not an intrinsic property of AFPs, and the interactions of INPs and different AFPs proceed through protein-specific rather than universal molecular mechanisms.

Synergy between Antifreeze Proteins Is Driven by Complementary Ice-Binding.

In some cold-adapted organisms, over a dozen isoforms of antifreeze (glyco)proteins or AF(G)Ps are present. Although these isoforms are structurally similar, their ability to inhibit ice growth varies significantly, and, in some fish, passive isoforms can be much more abundant than the active ones. Laboratory experiments demonstrated more than a decade ago that mixtures of AFP isoforms can exhibit synergistic enhancement of each other's activity. The mechanism of this synergy effect has remained obscure and is addressed here. Using cold-stages, microfluidics, and fluorescence microscopy, the activity of binary mixtures of structurally distinct AF(G)Ps from different fish and plant species was measured. While several mixtures exhibited enhancement, some mixtures exhibited antagonism. These latter mixtures included AF(G)Ps that bind to the same crystal planes, thereby exhibiting competition. Fluorescence microscopy experiments with a synergistic mixture of two isoform types labeled with different dyes showed they bound to different crystal planes. These results helped develop a kinetic description of the mechanism by which AF(G)Ps achieve synergy. The requirements of an active isoform include high adsorption rates, and prism plane binding, while passive isoforms usually bind to a pyramidal plane at slower rates. For synergy to occur, an active isoform first binds to the faster growing prism plane. This binding slows the advancement of the prism plane and creates more pyramidal surfaces to which a passive isoform bind. These results, in part, explain the biological observation of isoform distribution in fish, and the physical chemistry of the synergistic crystal growth inhibition by two inhibitors.

Effect of Antifreeze Glycoproteins on Organoid Survival during and after Hypothermic Storage.

We study the effect of antifreeze glycoproteins (AFGPs) on the survival of organoids under hypothermic conditions. We find that the survival of organoids in cold conditions depends on their developmental stage. Mature organoids die within 24 h when being stored at 4 °C, while cystic organoids can survive up to 48 h. We find that in the presence of AFGPs, the organoid survival is prolonged up to 72 h, irrespective of their developmental stage. Fluorescence microscopy experiments reveal that the AFGPs predominately localize at the cell surface and cover the cell membranes. Our findings support a mechanism in which the positive effect of AFGPs on cell survival during hypothermic storage involves the direct interaction of AFGPs with the cell membrane. Our research highlights organoids as an attractive multicellular model system for studying the action of AFGPs that bridges the gap between single-cell and whole-organ studies.

Determination of the Solution Structure of Antifreeze Glycoproteins Using Two-Dimensional Infrared Spectroscopy.

We study the solution structure of antifreeze glycoproteins (AFGPs) with linear and two-dimensional infrared spectroscopy (2D-IR). With 2D-IR, we study the coupling between the amide I and amide II vibrations of AFGPs. The measured nonlinear spectral response constitutes a much more clearly resolved amide I spectrum than the linear absorption spectrum of the amide I vibrations and allows us to identify the different structural elements of AFGPs in solution. We find clear evidence for the presence of polyproline II (PPII) helical structures already at room temperature, and we find that the fraction of PPII structures increases when the temperature is decreased to the biological working temperature of AFGP. We observe that inhibition of the antifreeze activity of AFGP using borate buffer or enhancing the antifreeze activity using sulfate buffer does not lead to significant changes in the protein conformation. This finding indicates that AFGPs bind to ice with their sugar side chains.

Antifreeze Glycoproteins Bind Irreversibly to Ice.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) inhibit ice growth via an adsorption-inhibition mechanism that assumes irreversible binding of AF(G)Ps to embryonic ice crystals and the inhibition of further growth. The irreversible binding of antifreeze glycoproteins (AFGPs) to ice has been questioned and remains poorly understood. Here, we used microfluidics and fluorescence microscopy to investigate the nature of the binding of small and large AFGP isoforms. We found that both AFGP isoforms bind irreversibly to ice, as evidenced by microfluidic solution exchange experiments. We measured the adsorption rate of the large AFGP isoform and found it to be 50% faster than that of AFP type III. We also found that the AFGP adsorption rate decreased by 65% in the presence of borate, a well-known inhibitor of AFGP activity. Our results demonstrate that the adsorption rate of AFGPs to ice is crucial for their ice growth inhibition capability.

Dynamics of the Hydration Water of Antifreeze Glycoproteins.

Antifreeze glycoproteins (AFGPs) are unique proteins that inhibit the growth of ice by a mechanism that is still unclear. We study the dynamics of water in aqueous solutions of small and large isoforms of AFGPs using polarization-resolved femtosecond infrared spectroscopy. We find that a fraction of the water molecules is strongly slowed down by the interaction with the antifreeze glycoprotein surface. The fraction of slow water molecules scales with the size and concentration of AFGP, and is similar to the fraction of slow water observed for nonantifreeze proteins, both at room temperature and close to biologically relevant working temperatures. We observe that inhibiting AFGP antifreeze activity using borate buffer induces no changes in the dynamics of water hydrating the AFGP. Our findings support a mechanism in which the sugar unit of AFGP forms the active ice-binding site.

Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature.

The remarkable adaptive strategies of insects to extreme environments are linked to the biochemical compounds in their body fluids. Trehalose, a versatile sugar molecule, can accumulate to high levels in freeze-tolerant and freeze-avoiding insects, functioning as a cryoprotectant and a supercooling agent. Antifreeze proteins (AFPs), known to protect organisms from freezing by lowering the freezing temperature and deferring the growth of ice, are present at high levels in some freeze-avoiding insects in winter, and yet, paradoxically are found in some freeze-tolerant insects. Here, we report a previously unidentified role for AFPs in effectively inhibiting trehalose precipitation in the hemolymph (or blood) of overwintering beetle larvae. We determine the trehalose level (29.6 ± 0.6 mg/mL) in the larval hemolymph of a beetle, Dendroides canadensis, and demonstrate that the hemolymph AFPs are crucial for inhibiting trehalose crystallization, whereas the presence of trehalose also enhances the antifreeze activity of AFPs. To dissect the molecular mechanism, we examine the molecular recognition between AFP and trehalose crystal interfaces using molecular dynamics simulations. The theory corroborates the experiments and shows preferential strong binding of the AFP to the fast growing surfaces of the sugar crystal. This newly uncovered role for AFPs may help explain the long-speculated role of AFPs in freeze-tolerant species. We propose that the presence of high levels of molecules important for survival but prone to precipitation in poikilotherms (their body temperature can vary considerably) needs a companion mechanism to prevent the precipitation and here present, to our knowledge, the first example. Such a combination of trehalose and AFPs also provides a novel approach for cold protection and for trehalose crystallization inhibition in industrial applications.

Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.

Investigation of the Ice-Binding Site of an Insect Antifreeze Protein Using Sum-Frequency Generation Spectroscopy.

We study the ice-binding site (IBS) of a hyperactive antifreeze protein from the beetle Dendroides canadensis (DAFP-1) using vibrational sum-frequency generation spectroscopy. We find that DAFP-1 accumulates at the air-water interface due to the hydrophobic character of its threonine-rich IBS while retaining its highly regular ß-helical fold. We observe a narrow band at 3485 cm(-1) that we assign to the O-H stretch vibration of threonine hydroxyl groups of the IBS. The narrow character of the 3485 cm(-1) band suggests that the hydrogen bonds between the threonine residues at the IBS and adjacent water molecules are quite similar in strength, indicating that the IBS of DAFP-1 is extremely well-ordered, with the threonine side chains showing identical rotameric confirmations. The hydrogen-bonded water molecules do not form an ordered ice-like layer, as was recently observed for the moderate antifreeze protein type III. It thus appears that the antifreeze action of DAFP-1 does not require the presence of ordered water but likely results from the direct binding of its highly ordered array of threonine residues to the ice surface.

Efficiency of antifreeze glycoproteins for cryopreservation of Nili-Ravi (Bubalus bubalis) buffalo bull sperm.

Experiments were conducted to evaluate the effect of Antarctic fish antifreeze glycoproteins, (AFGP) size 1-5 (34-10.5 kDa) and 7-8 (3.2 and 2.4 kDa) in extender on buffalo bull sperm at cooling (4 °C) and at post thawing. Semen was collected from three Nili-Ravi buffalo bulls with artificial vagina for 3 weeks. Qualifying ejaculates from each buffalo bull were diluted (at 37 °C having 50×10(6) sperm/mL) in tris-citric acid extender containing AFGP at 0 (control), 0.1, 1 and 10 µg/mL. An aliquot of diluted semen was evaluated for sperm progressive motility and plasma membrane integrity, while the remaining fraction was cooled to 4 °C in 2 h. Further, an aliquot of cooled semen was evaluated for the previously described variables and the remaining fraction was cryopreserved (-196 °C). After 24 h of storage, straws were thawed at 37 °C for 30 s to assess post-thaw sperm quality. Inclusion of AFGP in the extender did not affect (P>0.05) sperm progressive motility and plasma membrane integrity of buffalo bull sperm at cooling stage (4 °C). However, at post thawing, improvement (P<0.05) in sperm progressive motility and plasma membrane integrity was recorded in extender containing AFGP 1-5 and AFGP 7-8 at 1 µg/mL compared to the control. Percentage of live sperm with an intact acrosome remained similar (P>0.05) in extenders containing different amounts of AFGP and control. In conclusion, supplementation of 1 µg/ml of AFGP in extender improved the motility and plasma membrane integrity of Nili-Ravi buffalo sperm after thawing.

Variation in blood serum antifreeze activity of Antarctic Trematomus fishes across habitat temperature and depth.

High latitude waters in the Southern Ocean can be near their freezing point and remain ice-covered throughout the year whereas lower latitude Southern Ocean waters have seasonal ice coverage and comparatively large (6 °C) annual temperature changes. The genus Trematomus (suborder Notothenioidei) is regarded primarily as a high latitude group because of its abundance there, they also inhabit the warmer regions in smaller numbers. Freeze avoidance in the notothenioids is linked to the presence of two antifreeze proteins (AFPs); the antifreeze glycoproteins (AFGPs) and antifreeze potentiating protein (AFPP), both of which adsorb to internal ice crystals inhibiting growth. Both high and low latitude trematomids possess sufficient AFP to lower their blood freezing point below that of seawater (-1.9 °C). We investigated the contributions of AFGPs and AFPP to the blood freezing point depression to determine how they varied with depth, water temperature, and the presence of ice. High latitude trematomids had lower blood freezing points than those inhabiting lower latitude waters indicating differences in their freeze avoidance capacities. Lower freezing points were associated with higher levels of antifreeze activity due to higher levels of both AFGP and AFPP. Populations of Trematomus hansoni and Trematomus bernacchii from shallow depths appear more freeze avoidant than populations inhabiting deep, ice-free water based on their lower freezing points and higher antifreeze activities. Gel electrophoresis of the trichloroacetic acid-soluble AFGPs indicates that only high molecular weight isoforms, which contribute more to AFGP activity, vary across species as well as between individuals of a species.

Observation of ice-like water layers at an aqueous protein surface.

We study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0 °C to -2 °C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice.

Antifreeze protein-induced superheating of ice inside Antarctic notothenioid fishes inhibits melting during summer warming.

Antifreeze proteins (AFPs) of polar marine teleost fishes are widely recognized as an evolutionary innovation of vast adaptive value in that, by adsorbing to and inhibiting the growth of internalized environmental ice crystals, they prevent death by inoculative freezing. Paradoxically, systemic accumulation of AFP-stabilized ice could also be lethal. Whether or how fishes eliminate internal ice is unknown. To investigate if ice inside high-latitude Antarctic notothenioid fishes could melt seasonally, we measured its melting point and obtained a decadal temperature record from a shallow benthic fish habitat in McMurdo Sound, Antarctica. We found that AFP-stabilized ice resists melting at temperatures above the expected equilibrium freezing/melting point (eqFMP), both in vitro and in vivo. Superheated ice was directly observed in notothenioid serum samples and in solutions of purified AFPs, and ice was found to persist inside live fishes at temperatures more than 1 °C above their eqFMP for at least 24 h, and at a lower temperature for at least several days. Field experiments confirmed that superheated ice occurs naturally inside wild fishes. Over the long-term record (1999-2012), seawater temperature surpassed the fish eqFMP in most summers, but never exceeded the highest temperature at which ice persisted inside experimental fishes. Thus, because of the effects of AFP-induced melting inhibition, summer warming may not reliably eliminate internal ice. Our results expose a potentially antagonistic pleiotropic effect of AFPs: beneficial freezing avoidance is accompanied by melting inhibition that may contribute to lifelong accumulation of detrimental internal ice crystals.

Molecular recognition of methyl α-D-mannopyranoside by antifreeze (glyco)proteins.

Antifreeze proteins and glycoproteins [AF(G)Ps] have been well-known for more than three decades for their ability to inhibit the growth and recrystallization of ice through binding to specific ice crystal faces, and they show remarkable structural compatibility with specific ice crystal faces. Here, we show that the crystal growth faces of methyl α-D-mannopyranoside (MDM), a representative pyranose sugar, also show noteworthy structural compatibility with the known periodicities of AF(G)Ps. We selected fish AFGPs (AFGP8, AFGP1-5), and a beetle AFP (DAFP1) with increasing antifreeze activity as potential additives for controlling MDM crystal growth. Similar to their effects on ice growth, the AF(G)Ps can inhibit MDM crystal growth and recrystallization, and more significantly, the effectiveness for the AF(G)Ps are well correlated with their antifreeze activity. MDM crystals grown in the presence of AF(G)Ps are smaller and have better defined shapes and are of higher quality as indicated by single crystal X-ray diffraction and polarized microscopy than control crystals, but no new polymorphs of MDM were identified by single crystal X-ray diffraction, solid-state NMR, and attenuated total reflectance infrared spectroscopy. The observed changes in the average sizes of the MDM crystals can be related to the changes in the number of the MDM nuclei in the presence of the AF(G)Ps. The critical free energy change differences of the MDM nucleation in the absence and presence of the additives were calculated. These values are close to those of the ice nucleation in the presence of AF(G)Ps suggesting similar interactions are involved in the molecular recognition of MDM by the AF(G)Ps. To our knowledge this is the first report where AF(G)Ps have been used to control crystal growth of carbohydrates and on AFGPs controlling non-ice-like crystals. Our finding suggests MDM might be a possible alternative to ice for studying the detailed mechanism of AF(G)P-crystal interactions. The relationships between AF(G)Ps and carbohydrate binding proteins are also discussed. The structural compatibility between AF(G)Ps and growing crystal faces demonstrated herein adds to the repertoire of molecular recognition by AF(G)Ps, which may have potential applications in the sugar, food, pharmaceutical, and materials industries.

The role of sulfates on antifreeze protein activity.

In the present study, we have investigated the effect of sodium sulfate (Na2SO4) buffer on the antifreeze activity of DAFP-1, the primary AFP in the hemolymph of the beetle Dendroides canadensis. In contrast to previous studies, we found evidence that sodium sulfate does not suppress antifreeze activity of DAFP-1. Terahertz absorption spectroscopy (THz) studies were combined with molecular dynamics (MD) simulations to investigate the change in collective hydrogen bond dynamics in the vicinity of the AFP upon addition of sodium sulfate. The MD simulations revealed that the gradient of H-bond dynamics toward the ice-binding site is even more pronounced when adding sodium sulfate: The cosolute dramatically slows the hydrogen bond dynamics on the ice-binding plane of DAFP-1, whereas it has a more modest effect in the vicinity of other parts of the protein. These theoretical predictions are in agreement with the experimentally observed increase in THz absorption for solvated DAFP-1 upon addition of sodium sulfate. These studies support our previously postulated mechanism for AF activity, with a preferred ice binding by threonine on nanoice crystals which is supported by a long-range effect on hydrogen bond dynamics.

Chemical synthesis of a masked analogue of the fish antifreeze potentiating protein (AFPP).

A recently identified Antarctic fish protein termed antifreeze potentiating protein (AFPP) is thought to act as an adjunct to the previously characterised antifreeze glycoproteins (AFGPs), the two acting together to inhibit ice crystal growth in vivo. Elucidating the functional properties of the new AFPP requires access to large amounts of pure product, but the paucity of natural material necessitates alternative approaches. We therefore embarked on the total chemical synthesis of the AFPP, through a convergent ligation strategy. After many challenges, mostly due to the solubility issues of the peptide fragments, and several revisions of the original synthetic strategy, we have successfully synthesized a masked analogue of AFPP. The key to the successful synthesis was the use of a solubilising tag attached through a hydrolysable linker.

Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides.

Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation.