Biochemistry of fish antifreeze proteins

Four distinct macromolecular antifreezes have been isolated and characterized from different marine fish. These include the glycoprotein antifreezes (Mr 2.5-33 K), which are made up of a repeating tripeptide (Ala-Ala-Thr)n with a disaccharide attached to the threonyl residues, and three antifreeze protein (AFP) types. Type I is an alanine-rich, amphiphilic, alpha-helix (Mr 3-5 K); type II is a larger protein (Mr 14 K) with a high content of reverse turns and five disulfide bridges; and type III is intermediate in size (Mr 6-7 K) with no distinguishing features of secondary structure or amino acid composition. Despite their marked structural differences, all four antifreeze types appear to function in the same way by binding to the prism faces of ice crystals and inhibiting growth along the a-axes. It is suggested that type I AFP binds preferentially to the prism faces as a result of interactions between the helix macrodipole and the dipoles on the water molecules in the ice lattice. Binding is stabilized by hydrogen bonding, and the amphiphilic character of the helix results in the hydrophobic phase of the helix being exposed to the solvent. When the solution temperature is lowered further, ice crystal growth occurs primarily on the uncoated, unordered basal plane resulting in bipyramidal-shaped crystals. The structural features of type I AFP that could contribute to this mechanism of action are reviewed. Current challenges lie in solving the other antifreeze structures and interpreting them in light of what appears to be a common mechanism of action.     

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.     

昆虫低温生物学:Ⅱ.冰核物质(冰核蛋白)和昆虫的耐冻性

体系在低于熔点温度时才结冰的现象 ,叫过冷却 (supercooling)。体系开始结冰时的温度称为过冷却点 (supercooling point,SCP)。在适当的低温 ,体系内需存在一起始结冰的冰核 ,才能诱导冰晶产生 ,此物质称为冰核剂 (icenucleating agents,INA)。昆虫体内各腔室充满组织液 ,各腔室 (如消化系统和细胞内 )因所含 INA的冰核活性的不同 ,而使结冰的温度各异 ,所受低温伤害也不同。冰核常存在于昆虫血淋巴内 ,提高溶液的 SCP,降低其过冷却能力 ,引起胞外结冰。冰核物质的活性越高 ,SCP越高 ,虫体也能在较高的低温结冰。昆虫体内有不同性质…     

Systematic size study of an insect antifreeze protein and its interaction with ice

Because of their remarkable ability to depress the freezing point of aqueous solutions, antifreeze proteins (AFPs) play a critical role in helping many organisms survive subzero temperatures. The beta-helical insect AFP structures solved to date, consisting of multiple repeating circular loops or coils, are perhaps the most regular protein structures discovered thus far. Taking an exceptional advantage of the unusually high structural regularity of insect AFPs, we have employed both semiempirical and quantum mechanics computational approaches to systematically investigate the relationship between the number of AFP coils and the AFP-ice interaction energy, an indicator of antifreeze activity. We generated a series of AFP models with varying numbers of 12-residue coils (sequence TCTxSxxCxxAx) and calculated their interaction energies with ice. Using several independent computational methods, we found that the AFP-ice interaction energy increased as the number of coils increased, until an upper bound was reached. The increase of interaction energy was significant for each of the first five coils, and there was a clear synergism that gradually diminished and even decreased with further increase of the number of coils. Our results are in excellent agreement with the recently reported experimental observations.     

Purification of antifreeze proteins by adsorption to ice

Antifreeze proteins (AFPs) can protect organisms from freezing injury by adsorbing to ice and inhibiting its growth. We describe here a method where ice, grown on a cold finger, is used to selectively adsorb and purify these ice-binding proteins from a crude mixture. Type III recombinant AFP was enriched approximately 50-fold after one round of partitioning into ice and purified to homogeneity by a second round. This method can also be used to purify non-ice-binding proteins by linkage to AFP domains as demonstrated by the recovery of a 50 kDa maltose-binding protein-AFP fusion from a crude lysate of Escherichia coli.     

昆虫抗冻蛋白的分离纯化及特性分析

昆虫抗冻蛋白具有很高的热滞活性,可保护机体免受结冰引起的伤害。昆虫抗冻蛋白的分离纯化多采用凝胶过滤层析、离子交换层析及HPLC等技术,已用于鱼类抗冻蛋白纯化的冰亲和纯化(IAP)技术也可考虑应用于昆虫抗冻蛋白的分离提纯。昆虫抗冻蛋白具有高活性,规则的一级结构及类似的冰晶结合表面等特性。     

Adsorption to ice of fish antifreeze glycopeptides 7 and 8.

Experimental results show that fish antifreeze glycopeptides (AFGPs) 8 and 7 (with 4 and 5 repeats respectively of the Ala-Ala-Thr backbone sequence) bond onto ice prism planes aligned along a-axes, and inhibit crystal growth on prism planes and on surfaces close to that orientation. The 9.31-A repeat spacing of the AFGP in the polyproline II helix configuration, deduced from NMR studies, matches twice the repeat spacing of ice in the deduced alignment direction, 9.038 A, within 3%. A specific binding model is proposed for the AFGP and for the alpha-helical antifreeze peptide of winter flounder. For AFGP 7-8, two hydroxyl groups of each disaccharide (one disaccharide is attached to each threonine) reside within the ice surface, so that they are shared between the ice crystal and the disaccharide. This provides 24 hydrogen bonds between AFGP 8 and the ice and 30 for AFGP 7, explaining why the chemical adsorption is virtually irreversible and the crystal growth can be stopped virtually completely. The same scheme of sharing polar groups with the ice works well with the alpha-helical antifreeze of winter flounder, for which an amide as well as several hydroxyls are shared. The sharing of polar groups with the ice crystal, rather than hydrogen-bonding to the ice surface, may be a general requirement for adsoprtion-inhibition of freezing.     

Epithelial dominant expression of antifreeze proteins in cunner suggests recent entry into a high freeze-risk ecozone

Most marine teleost fishes residing in a high freeze-risk ecozone, such as the coastal waters of Newfoundland during winter, avoid freezing by secreting high concentrations of antifreeze proteins (AFP) into their blood plasma where they can bind to and prevent the growth of ice that enter the fish. Cunner (Tautogolabrus adspersus), which overwinter in such shallow waters are the only known exception. Although this species does produce type I AFP, the plasma levels are too low to be of value as a freeze protectant. Southern and Northern blot analyses carried out in this study establish that the cunner AFP genes belong to a multigene family that is predominantly expressed in external epithelia (skin and gill filaments). These results support the hypothesis that the survival of cunner in icy waters is attributable in part to epithelial AFP that help block ice propagation into their interior milieu. In contrast to the cunner, heterospecifics occupying the same habitat have greater freeze protection because they produce AFP in the liver for export to the plasma as well as in external epithelia. Since the external epithelia would be the first tissue to come into contact with ice it is possible that one of the earliest steps involved in the evolution of freeze resistant fish could have been the expression of AFP in tissues such as the skin. We suggest that this epithelial-dominant AFP expression represents a primitive stage in AFP evolution and propose that cunner began to inhabit “freeze-risk ecozones” more recently than heterospecifics.     

SMR proteins SugE and EmrE bind ligand with similar affinity and stoichiometry

Suppressor of a groEL mutation protein E (SugE) is a small multidrug resistance (SMR) homologue. In comparison with other SMR proteins, SugE promotes bacterial resistance to a narrow range of quaternary ammonium compounds (QACs). Isothermal titration calorimetry was used to study the binding of QACs to Escherichia coli SugE in different membrane mimetic environments. In this study, the binding stoichiometry of SugE to drug was found to be 1:1, and the binding of SugE to drug was observed with the dissociation constant (K(D)) in the micromolar range for each of the drugs in the membrane mimetic environments explored. This interaction appears to be enthalpy-driven with enthalpies of 8-12 kcal/mol for each of the drugs. These results are similar to those found with drug binding to the SMR protein EmrE in an earlier study.     

Diffusion NMR studies on fish antifreeze proteins and synthetic analogues

Pulsed field gradient spin echo NMR spectroscopy was used to measure diffusion coefficients of the alpha-helical type I antifreeze protein from the winter flounder, two synthetic derivatives in which the four Thr residues were replaced with Val and Ala, respectively, and the low molecular weight fraction antifreeze glycoprotein. Under the conditions studied, the natural type I antifreeze protein and low molecular weight glycoprotein gave diffusion values that were consistent with the presence of monomeric protein in solution. While significant aggregation of the Ala analogue was observed (2-10 mM), there was no evidence for aggregation in the Val analogue (1-3 mM). These results are compared with previously reported solubility and thermal hysteresis data and the implications for the design of synthetic antifreeze proteins are discussed.     

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains

Using synthetic DNA, we assembled a gene encoding a protein identical in sequence to one of the antifreeze proteins produced by the fish Pseudopleuronectes americanus (winter flounder). To address the relationship between structure and function, we also assembled genes encoding proteins varying in sequence and length. The synthetic genes were cloned into a bacterial expression vector to generate translational fusions to the 3' end of a truncated staphylococcal protein A gene; the chimeric proteins encoded by these fusions, varying only in their antifreeze domains, were isolated from Escherichia coli. The antifreeze domains conferred the ability to inhibit ice recrystallization, which is characteristic of naturally occurring antifreeze proteins, on the chimeric proteins. The chimeric proteins varied in their effectiveness of inhibiting ice recrystallization according to the number of 11-amino acid repeats present in the antifreeze moiety. A protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four, and five repeats. Some activity was lost upon removal of either the salt bridge or the carboxyl-terminal arginine, but surprisingly, not when both features were absent together.     

Understanding the mechanism of ice binding by type III antifreeze proteins

Type III antifreeze proteins (AFPs) are present in the body fluids of some polar fishes where they inhibit ice growth at subzero temperatures. Previous studies of the structure of type III AFP by NMR and X-ray identified a remarkably flat surface on the protein containing amino acids that were demonstrated to be important for interaction with ice by mutational studies. It was proposed that this protein surface binds onto the (1 0 [\bar 1] 0) plane of ice with the key amino acids interacting directly with the water molecules in the ice crystal. Here, we show that the mechanism of type III AFP interaction with ice crystals is more complex than that proposed previously. We report a high-resolution X-ray structure of type III AFP refined at 1.15 A resolution with individual anisotropic temperature factors. We report the results of ice-etching experiments that show a broad surface coverage, suggesting that type III AFP binds to a set of planes that are parallel with or inclined at a small angle to the crystallographic c-axis of the ice crystal. Our modelling studies, performed with the refined structure, confirm that type III AFP can make energetically favourable interactions with several ice surfaces.     

Contribution of hydrophobic residues to ice binding by fish type III antifreeze protein

Type III antifreeze protein (AFP) is a 7-kDa globular protein with a flat ice-binding face centered on Ala 16. Neighboring hydrophilic residues Gln 9, Asn 14, Thr 15, Thr 18 and Gln 44 have been implicated by site-directed mutagenesis in binding to ice. These residues have the potential to form hydrogen bonds with ice, but the tight packing of side chains on the ice-binding face limits the number and strength of possible hydrogen bond interactions. Recent work with alpha-helical AFPs has emphasized the hydrophobicity of their ice-binding sites and suggests that hydrophobic interactions are important for antifreeze activity. To investigate the contribution of hydrophobic interactions between type III AFP and ice, Leu, Ile and Val residues on the rim of the ice-binding face were changed to alanine. Mutant AFPs with single alanine substitutions, L19A, V20A, and V41A, showed a 20% loss in activity. Doubly substituted mutants, L19A/V41A and L10A/I13A, had less than 50% of the activity of the wild type. Thus, side chain substitutions that leave a cavity or undercut the contact surface are almost as deleterious to antifreeze activity as those that lengthen the side chain. These mutations emphasize the importance of maintaining a specific surface contour on the ice-binding face for docking to ice.     

Insight into the binding of antifreeze proteins to ice surfaces via 13C spin lattice relaxation solid-state NMR

The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of alpha-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP's ice-binding side, a variable-temperature dependent (13)C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain (13)C labeled HPLC6 isoforms of the type I AFPs each frozen in H(2)O and D(2)O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the alpha-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T(1) data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the alpha-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface.     

Fluorescence microscopy evidence for quasi-permanent attachment of antifreeze proteins to ice surfaces

Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins.     

Direct visualization of spruce budworm antifreeze protein interacting with ice crystals: basal plane affinity confers hyperactivity

Antifreeze proteins (AFPs) protect certain organisms from freezing by adhering to ice crystals, thereby preventing their growth. All AFPs depress the nonequilibrium freezing temperature below the melting point; however AFPs from overwintering insects, such as the spruce budworm (sbw) are 10-100 times more effective than most fish AFPs. It has been proposed that the exceptional activity of these AFPs depends on their ability to prevent ice growth at the basal plane. To test the hypothesis that the hyperactivity of sbwAFP results from direct affinity to the basal plane, we fluorescently tagged sbwAFP and visualized it on the surface of ice crystals using fluorescence microscopy. SbwAFP accumulated at the six prism plane corners and the two basal planes of hexagonal ice crystals. In contrast, fluorescently tagged fish type III AFP did not adhere to the basal planes of a single-crystal ice hemisphere. When ice crystals were grown in the presence of a mixture of type III AFP and sbwAFP, a hybrid crystal shape was produced with sbwAFP bound to the basal planes of truncated bipyramidal crystals. These observations are consistent with the blockage of c-axial growth of ice as a result of direct interaction of sbwAFP with the basal planes.