Solution structure of a recombinant type I sculpin antifreeze protein

We have determined the solution structure of rSS3, a recombinant form of the type I shorthorn sculpin antifreeze protein (AFP), at 278 and 268 K. This AFP contains an unusual sequence of N-terminal residues, together with two of the 11-residue repeats that are characteristic of the type I winter flounder AFP. The solution conformation of the N-terminal region of the sculpin AFP has been assumed to be the critical factor that results in recognition of different ice planes by the sculpin and flounder AFPs. At 278 K, the two repeats units (residues 11-20 and 21-32) in rSS3 form a continuous alpha-helix, with the residues 30-33 in the second repeat somewhat less well defined. Within the N-terminal region, residues 2-6 are well defined and helical and linked to the main helix by a more flexible region comprising residues A7-T11. At 268 K the AFP is overall more helical but retains the apparent hinge region. The helical conformation of the two repeats units is almost identical to the corresponding repeats in the type I winter flounder AFP. We also show that while tetracetylated rSS3 has antifreeze activity comparable to the natural AFP, its overall structure is the same as that of the unacetylated peptide. These data provide some insight into the structural determinants of antifreeze activity and should assist in the development of models that explain the recognition of different ice interfaces by the sculpin and flounder type I AFPs.     

A re-evaluation of the role of type IV antifreeze protein

A lipoprotein-like antifreeze protein (type IV AFP) has previously been isolated only from the blood plasma of the longhorn sculpin. However, the plasma antifreeze activity in all individuals of this species tested from Newfoundland and New Brunswick waters ranges from low to undetectable. A close relative of the longhorn sculpin, the shorthorn sculpin, does have appreciable antifreeze activity in its blood but this is virtually all accounted for by the α-helical, alanine-rich type I AFP, other isoforms of which are also present in the skin of both fishes. We have characterized a putative ortholog of type IV AFP in shorthorn sculpin by cDNA cloning. This 12.2-kDa Gln-rich protein is 87% identical to the longhorn sculpin’s type IV AFP. Recombinant versions of both orthologs were produced in bacteria and shown to have antifreeze activity. Immunoblotting with antibodies raised to type IV AFP shows this protein present in longhorn sculpin plasma at levels of less than 100 μg/mL, which are far too low to protect the blood from freezing at the temperature of icy seawater. This confirms the results of direct antifreeze assays on the plasmas. It appears that type IV AFP has the potential to develop as a functional antifreeze in these fishes but may not have been selected for this role because of the presence of type I AFP. Consistent with this hypothesis is the observation that the type IV AFP gene has not been amplified the way functional antifreeze protein genes have in all other species examined.     

A re-evaluation of the role of type IV antifreeze protein

A lipoprotein-like antifreeze protein (type IV AFP) has previously been isolated only from the blood plasma of the longhorn sculpin. However, the plasma antifreeze activity in all individuals of this species tested from Newfoundland and New Brunswick waters ranges from low to undetectable. A close relative of the longhorn sculpin, the shorthorn sculpin, does have appreciable antifreeze activity in its blood but this is virtually all accounted for by the α-helical, alanine-rich type I AFP, other isoforms of which are also present in the skin of both fishes. We have characterized a putative ortholog of type IV AFP in shorthorn sculpin by cDNA cloning. This 12.2-kDa Gln-rich protein is 87% identical to the longhorn sculpin’s type IV AFP. Recombinant versions of both orthologs were produced in bacteria and shown to have antifreeze activity. Immunoblotting with antibodies raised to type IV AFP shows this protein present in longhorn sculpin plasma at levels of less than 100 μg/mL, which are far too low to protect the blood from freezing at the temperature of icy seawater. This confirms the results of direct antifreeze assays on the plasmas. It appears that type IV AFP has the potential to develop as a functional antifreeze in these fishes but may not have been selected for this role because of the presence of type I AFP. Consistent with this hypothesis is the observation that the type IV AFP gene has not been amplified the way functional antifreeze protein genes have in all other species examined.     

Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes.

The noncolligative peptide and glycopeptide antifreezes found in some cold-water fish act by binding to the ice surface and preventing crystal growth, not by altering the equilibrium freezing point of the water. A simple crystal growth and etching technique allows determination of the crystallographic planes where the binding occurs. In the case of elongated molecules, such as the alpha-helical peptides in this report, it also allows a deduction of the molecular alignment on the ice surface. The structurally similar antifreeze peptides from winter flounder (Pseudopleuronectes americanus) and Alaskan plaice (Pleuronectes quadritaberulatus) adsorb onto the (2021) pyramidal planes of ice, whereas the sculpin (Myoxocephalus scorpius) peptide adsorbs on (2110), the secondary prism planes. All three are probably aligned along (0112). These antifreeze peptides have 11-amino acid sequence repeats ending with a polar residue, and each repeat constitutes a distance of 16.5 A along the helix, which nearly matches the 16.7 A repeat spacing along (0112) in ice. This structural match is undoubtedly important, but the mechanism of binding is not yet clear. The suggested mechanism of growth inhibition operates through the influence of local surface curvature upon melting point and results in complete inhibition of the crystal growth even though individual antifreeze molecules bind at only one interface orientation.     

A model for binding of an antifreeze polypeptide to ice.

A model is proposed, based on recent peptide analog and ice crystal etching studies, whereby an alanine-rich, alpha-helical antifreeze polypeptide (AFP) from the winter flounder inhibits the growth of ice crystals by hydrogen bonding of Thr, Asn, and Asp side chains in a specific pattern to the [2021] hexagonal bipyramidal planes of ice. It is further suggested that this mode of binding is unidirectional, maximizing opportunities for packing of AFPs on the ice surface, and that ice crystal growth inhibition occurs by a two-step mechanism involving hydrogen bonding and hydrophobic interpeptide interactions.     

NMR analysis of type III antifreeze protein intramolecular dimer. Structural basis for enhanced activity

The structure of a new antifreeze protein (AFP) variant, RD3, from antarctic eel pout (Rhigophila dearborni) with enhanced activity has been determined for the first time by nuclear magnetic resonance spectroscopy. RD3 comprises a unique translational topology of two homologous type III AFP globular domains, each containing one flat, ice binding plane. The ice binding plane of the N domain is located approximately 3.5 A "behind" that of the C domain. The two ice binding planes are located laterally with an angle of 32 +/- 12 degrees between the planes. These results suggest that the C domain plane of RD3 binds first to the ice [1010] prism plane in the <0001> direction, which induces successive ice binding of the N domain in the <0101> direction. This manner of ice binding caused by the unique structural topology of RD3 is thought to be crucial for the significant enhancement of antifreeze activity, especially at low AFP concentrations.     

Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations AFGP antifreeze glycoprotein(s) - AFP antifreeze polypeptide(s) - HPLC high performance liquid chromatography - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Structures of shorthorn sculpin antifreeze polypeptides

The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly alpha-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The alpha-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.     

Type I Antifreeze Proteins: Possible Origins from Chorion and Keratin Genes in Atlantic Snailfish

Type I antifreeze proteins (AFPs) are alanine-rich α-helical polypeptides found in some species of right-eye flounders, sculpin, and snailfish. In this study, a shorthorn sculpin skin type I cDNA clone was used to probe an Atlantic snailfish liver cDNA library in order to locate expressed genes corresponding to snailfish plasma AFPs. Clones isolated from the cDNA library had sections with substantial amino acid and nucleotide sequence similarity to snailfish type I AFPs. However, further analysis revealed that the positives were actually three different liver-expressed proteins—two were eggshell proteins, while the third was a type II keratin. We propose that a shift in reading frame could produce alanine-rich candidate AFPs with possible antifreeze activity or ice crystal modification properties. Furthermore, it is plausible that one or more of the liver-expressed proteins represent the progenitors of snailfish type I AFPs. [Reviewing Editor: Dr. John Oakeshott]     

Ice-binding mechanism of winter flounder antifreeze proteins

We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic effect play in ice binding are also highlighted. For the latter it is demonstrated that the surface of ice has a clathratelike structure which favors the partitioning of hydrophobic groups to the surface of ice. It is suggested that mutations that involve the deletion of hydrophobic residues (e.g., the Leu residues) will provide insight into the role the hydrophobic effect plays in partitioning these peptides to the surface of ice.     

Why does insect antifreeze protein from Tenebrio molitor produce pyramidal ice crystallites?

The antifreeze protein (AFP) reduces the growth rates of the ice crystal facets. In that process the ice morphology undergoes a modification. An AFP-induced surface pinning mechanism, through matching of periodic bond chains in two dimensions, enables two-dimensional regular ice-binding surfaces (IBSs) of the insect AFPs to engage a certain class of ice surfaces, called primary surfaces. They are kinetically stable surfaces with unambiguous and predetermined orientations. In this work, the orientations and molecular compositions of the primary ice surfaces that undergo growth rate reduction by the insect AFPs are obtained from first principles. Besides the basal face and primary prism, the ice surfaces engaged by insect AFPs include the specific ice pyramids produced by the insect AFP Tenebrio molitor (TmAFP). TmAFP-induced pyramids differ fundamentally from the ice pyramids produced by fish AFPs and antifreeze protein glycoproteins (AFPGs) as regards the ice surface configurations and the mode of interaction with the protein IBS. The molecular compositions of the TmAFP-induced pyramids are strongly bonded in two dimensions and have the constant face indices (101). In contrast, the molecular composition of the ice pyramids produced by fish AFPs and AFPGs are strongly bonded in only one direction and have variable face indices (h 0 l), none of which equal (101). The thus far puzzling behavior of the TmAFP in producing pyramidal crystallites is fully explained in agreement with experiment.     

Energy-optimized structure of antifreeze protein and its binding mechanism.

A combination of Monte Carlo simulated annealing and energy minimization was utilized to determine the conformation of the antifreeze protein from the fish winter flounder. It was found from the energy-optimized structure that the hydroxyl groups of its four threonine residues, i.e. Thr2, Thr13, Thr24, Thr35, are aligned on almost the same line parallel to the helix axis and separated successively by 16.1, 16.0 and 16.2 A, respectively, very close to the 16.6 A repeat spacing along [0112] in ice. Based on such a space match, a zipper-like model is proposed to elucidate the binding mechanism of the antifreeze protein to ice crystals. According to the current model, the antifreeze protein may bind to an ice nucleation structure in a zipper-like fashion through hydrogen bonding of the hydroxyl groups of these four Thr residues to the oxygen atoms along the [0112] direction in ice lattice, subsequently stopping or retarding the growth of ice pyramidal planes so as to depress the freeze point. The calculated results and the binding mechanism thus derived accord with recent experimental observations. The mechanistic implications derived from such a special antifreeze molecule might be generally applied to elucidate the structure-function relationship of other antifreeze proteins with the following two common features: (1) recurrence of a Thr residue (or any other polar amino acid residue whose side-chain can form a hydrogen bond with water) in an 11-amino-acid period along the sequence concerned; and (2) a high percentage of Ala residue component therein. Further experiments are suggested to test the ice binding model.     

Isolation and characterization of skin-type, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus octodecemspinosus

The antifreeze polypeptides (AFPs) are found in several marine fish and have been grouped into four distinct biochemical classes (type I-IV). Recently, the new subclass of skin-type, type I AFPs that are produced intracellularly as mature polypeptides have been identified in the winter flounder (Pleuronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius). This study demonstrates the presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs. Using polymerase chain reaction-based methods, a clone that encoded for a type I AFP was identified. The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol. A recombinant protein was produced in Escherichia coli and antifreeze activity was characterized. Four individual Ala-rich polypeptides with antifreeze activity were isolated from the skin tissue. One polypeptide was completely sequenced by tandem MS. This study provides the first evidence of a fish species that produces two different biochemical classes of antifreeze proteins (type I and type IV), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.     

Structure and function of antifreeze proteins

High-resolution three-dimensional structures are now available for four of seven non-homologous fish and insect antifreeze proteins (AFPs). For each of these structures, the ice-binding site of the AFP has been defined by site-directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice-binding proteins shows that they have the following attributes in common. The binding sites are relatively flat and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic -- more so than that portion of the protein exposed to the solvent. Surface-surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts.     

Quantitative and qualitative analysis of type III antifreeze protein structure and function

Some cold water marine fishes avoid cellular damage because of freezing by expressing antifreeze proteins (AFPs) that bind to ice and inhibit its growth; one such protein is the globular type III AFP from eel pout. Despite several studies, the mechanism of ice binding remains unclear because of the difficulty in modeling the AFP-ice interaction. To further explore the mechanism, we have determined the x-ray crystallographic structure of 10 type III AFP mutants and combined that information with 7 previously determined structures to mainly analyze specific AFP-ice interactions such as hydrogen bonds. Quantitative assessment of binding was performed using a neural network with properties of the structure as input and predicted antifreeze activity as output. Using the cross-validation method, a correlation coefficient of 0.60 was obtained between measured and predicted activity, indicating successful learning and good predictive power. A large loss in the predictive power of the neural network occurred after properties related to the hydrophobic surface were left out, suggesting that van der Waal's interactions make a significant contribution to ice binding. By combining the analysis of the neural network with antifreeze activity and x-ray crystallographic structures of the mutants, we extend the existing ice-binding model to a two-step process: 1) probing of the surface for the correct ice-binding plane by hydrogen-bonding side chains and 2) attractive van der Waal's interactions between the other residues of the ice-binding surface and the ice, which increases the strength of the protein-ice interaction.     

Type I shorthorn sculpin antifreeze protein: recombinant synthesis, solution conformation, and ice growth inhibition studies

A number of structurally diverse classes of "antifreeze" proteins that allow fish to survive in sub-zero ice-laden waters have been isolated from the blood plasma of cold water teleosts. However, despite receiving a great deal of attention, the one or more mechanisms through which these proteins act are not fully understood. In this report we have synthesized a type I antifreeze polypeptide (AFP) from the shorthorn sculpin Myoxocephalus scorpius using recombinant methods. Construction of a synthetic gene with optimized codon usage and expression as a glutathione S-transferase fusion protein followed by purification yielded milligram amounts of polypeptide with two extra residues appended to the N terminus. Circular dichroism and NMR experiments, including residual dipolar coupling measurements on a 15N-labeled recombinant polypeptide, show that the polypeptides are alpha-helical with the first four residues being more flexible than the remainder of the sequence. Both the recombinant and synthetic polypeptides modify ice growth, forming facetted crystals just below the freezing point, but display negligible thermal hysteresis. Acetylation of Lys-10, Lys-20, and Lys-21 as well as the N terminus of the recombinant polypeptide gave a derivative that displays both thermal hysteresis (0.4 degrees C at 15 mg/ml) and ice crystal faceting. These results confirm that the N terminus of wild-type polypeptide is functionally important and support our previously proposed mechanism for all type I proteins, in which the hydrophobic face is oriented toward the ice at the ice/water interface.     

Antifreeze protein-induced morphological modification mechanisms linked to ice binding surface

The mechanisms by which the antifreeze protein (AFP) modifies the ice morphology are identified precisely as surface poisoning by the ice binding surface (IBS) of insect AFPs and as bridge-induced surface reconstruction by the IBS of fish AFPs and antifreeze glycoproteins. The primary surfaces of hexagonal ice have predetermined face indices. The "two-dimensional" insect type IBS has regularly spaced binding intervals in two directions. It causes surface poisoning by matching and reinforcing simultaneously intersecting strong bonding directions on the primary ice surfaces. The secondary ice surfaces have variable face indices. The "one-dimensional" and "irregular" IBS variants of fish AFPs and antifreeze glycoproteins are either linearly extended with regular ice binding intervals or have ice binding sites lacking spacing regularity. These variants can bridge transversely lattice periods or shorter oxygen-oxygen distances between parallel adjacent strong bonding directions that do not intersect. Thus, one-dimensional and irregular IBS variants induce supplementary bridges cross-wise on selected secondary surfaces by mimicking strong bonding directions that are not present in the ice structure. These proteins cause surfaces with variable face indices, which in the absence of the AFPs would not grow flat, to appear in the morphology. Whereas for the primary ice surfaces it is only the morphological importance that is determined by the experimental conditions, for the secondary ice surfaces it is the face indices themselves that become adjusted in the process of maximizing the AFP-substrate interaction through attainment of the best structural match. The growth morphology of the AFP-ice system is derived from various factors, including the face indices, surface molecular compositions, relative growth rates, and the mechanisms responsible for that morphology. The theoretical formulation agrees with experiments over a wide range and resolves these, to date, unexplained phenomena.     

Structure-function relationship in a winter flounder antifreeze polypeptide. II. Alteration of the component growth rates of ice by synthetic antifreeze polypeptides

Using synthetic analogs of an alpha-helical winter flounder antifreeze polypeptide (AFP) we investigated some important molecular details of the mechanism of action of this AFP. Of the seven peptides synthesized, all but one were amino-terminal deletions of the native AFP. Three of the seven synthetic analogs possessed the same antifreeze activity as the native polypeptide; the other analogs were devoid of antifreeze activity. The growth rates along the a and c axes of ice in solutions of varying concentrations of the three active AFP analogs were examined. The a axis growth rates of ice were inversely proportional to the concentration of the active peptides. The c-axis growth rates of ice were also dependent on peptide concentration. The active peptides enhanced c-axis growth at lower concentrations, while at higher concentrations they inhibited c axis growth. The ability of the peptides to develop antifreeze activity and to alter the a and c axis growth rates of ice depended on the presence of appropriately positioned amino acid residues with hydrogen bonding side chains. From these observations we propose that at low concentrations the AFP, through dipolar interactions and hydrogen bonding, interact with the prism faces of ice retarding a axis growth. At these concentrations, the electrical field of the AFP helix-dipole, like an externally applied field (Bartlett, J.T., van der Heuval, A.P., and Mason, B.J. (1963) Z. Angew. Math. Phys. 14, 599-610), can enhance ice c axis growth. At higher concentrations, the AFP interact with all ice crystal planes and retard both a and c axis growth.     

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.     

Analysis of ice-binding sites in fish type II antifreeze protein by quantum mechanics

Many organisms living in cold environments can survive subzero temperatures by producing antifreeze proteins (AFPs) or antifreeze glycoproteins. In this paper we investigate the ice-binding surface of type II AFP by quantum mechanical methods, which, to the best of our knowledge, represents the first time that molecular orbital computational approaches have been applied to AFPs. Molecular mechanical approaches, including molecular docking, energy minimization, and molecular dynamics simulation, were used to obtain optimal systems for subsequent quantum mechanical analysis. We selected 17 surface patches covering the entire surface of the type II AFP and evaluated the interaction energy between each of these patches and two different ice planes using semi-empirical quantum mechanical methods. We have demonstrated the weak orbital overlay phenomenon and the change of bond orders in ice. These results consistently indicate that a surface patch containing 19 residues (K37, L38, Y20, E22, Y21, I19, L57, T56, F53, M127, T128, F129, R17, C7, N6, P5, G10, Q1, and W11) is the most favorable ice-binding site for both a regular ice plane and an ice plane where water O atoms are randomly positioned. Furthermore, for the first time the computation results provide new insights into the weakening of the ice lattice upon AFP binding, which may well be a primary factor leading to AFP-induced ice growth inhibition.