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.     

Rational design of alpha-helical antifreeze peptides.

The alanine-rich alpha-helical antifreeze protein from the winter flounder Pseudopleuronectes americanus adsorbs to specific planes of ice guided by an ice lattice match to threonine residues regularly spaced 16.6 A apart. We report here that by redesigning the winter flounder antifreeze peptide to incorporate a 27.1-A spacing between putative 'ice-binding' threonines, the deduced binding alignment of the helical molecule on the ice lattice is changed from the Miller indices directional vector [1102 ] to [2203 ]. Subsequent ice-binding characteristics are altered, including changes in adsorption specificity, decreases in thermal hysteresis activity and the formation of rotated hexagonal bipyramid ice crystal morphology.     

Valine substituted winter flounder ‘antifreeze': preservation of ice growth hysteresis

Three mutant polypeptides of the type I 37-residue winter flounder ‘antifreeze' protein have been synthesized. All four threonine residues in the native peptide were been mutated to serine, valine and glycine respectively and two additional salt bridges were incorporated into the sequences in order to improve aqueous solubility. The peptides were analyzed by nanoliter osmometry, the ‘ice hemisphere' test, the ‘crystal habit' test, measurement of ice growth hysteresis and CD spectroscopy. While the valine and serine mutants retain the α-helical structure, only the valine mutant retains ‘antifreeze' activity similar to that of the native protein. These data show that the threonine hydroxyl groups do not play a crucial role in the accumulation of the native ‘antifreeze' protein at the ice/water interface and the inhibition of ice growth below the equilibrium melting temperature.     

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.     

Increased flexibility decreases antifreeze protein activity

Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single α-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg37 HPLC6, nonamidated HPLC6 Ala37, amidated HPLC6 Ala37, and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still α-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface.     

Antifreeze proteins at the ice/water interface: three calculated discriminating properties for orientation of type I proteins

Antifreeze proteins (AFPs) protect many plants and organisms from freezing in low temperatures. Of the different AFPs, the most studied AFP Type I from winter flounder is used in the current computational studies to gain molecular insight into its adsorption at the ice/water interface. Employing molecular dynamics simulations, we calculate the free energy difference between the hydrophilic and hydrophobic faces of the protein interacting with ice. Furthermore, we identify three properties of Type I "antifreeze" proteins that discriminate among these two orientations of the protein at the ice/water interface. The three properties are: the "surface area" of the protein; a measure of the interaction of the protein with neighboring water molecules as determined by the number of hydrogen bond count, for example; and the side-chain orientation angles of the threonine residues. All three discriminants are consistent with our free energy results, which clearly show that the hydrophilic protein face orientations toward the ice/water interface, as hypothesized from experimental and ice/vacuum simulations, are incorrect and support the hypothesis that the hydrophobic face is oriented toward the ice/water interface. The adsorption free energy is calculated to be 2-3 kJ/mol.     

Analysis of shorthorn sculpin antifreeze protein stereospecific binding to (2-1 0) faces of ice.

In this paper we report the results of our studies on the stereospecific binding of shorthorn sculpin antifreeze protein (AFP) to (2 -1 0) secondary prism faces of ice. Using ice crystal growth and etching techniques together with molecular modeling, molecular dynamics, and energy minimization, we explain the nature of preferential binding of shorthorn sculpin AFP along the [1 2 2] direction on (2- 1 0) planes. In agreement with ice etching studies, the mechanism of preferential binding suggested by molecular modeling explains why the binding of shorthorn sculpin AFP occurs along [1 2 2] and not along its mirror symmetry-related direction [-1 -2 2] on (2 -1 0). This binding mechanism is based on the protein-crystal surface enantioselective recognition that utilizes both alpha-helical protein backbone matching to the (2 -1 0) surface topography and matching of side chains of polar/charged residues with specific water molecule positions in the ice surface. The mechanisms of winter flounder and shorthorn sculpin antifreeze binding to ice are compared.     

Hydrophobic analogues of the winter flounder 'antifreeze' protein

The synthesis, solution conformation and ice-growth inhibition properties of four new analogues of the type I 37-residue winter flounder 'antifreeze' protein are reported. All four analogues contain two extra salt bridges to facilitate comparison of results with previously published data. In two analogues, all four threonine residues in the native polypeptide were mutated to 2-amino butyric acid (an unnatural amino acid) and isoleucine, respectively. The butyric acid analogue was approximately 85% helical at 3 degrees C, modified the shape of ice growth, and exhibited reduced hysteresis compared to the native protein (9% at 4 mM). These results show that the gamma-methyl group of threonine, which is present in the sidechain of 2-amino butyric acid, is not sufficient for activity. The isoleucine analogue, in which the threonine hydroxyl group is replaced by an ethyl group, was 100% helical at 3 degrees C, showed no hysteresis but was able to modify the shape of ice crystal growth. In the third and fourth analogues, mutations of the aspartic acids 1 and 5 to alanine, and asparagines 16 and 27 to leucine in the threonine- and valine-substituted analogues did not affect the helicity of the polypeptides, but removed the ability to inhibit ice growth.     

Antifreeze protein from shorthorn sculpin: Identification of the ice-binding surface

Shorthorn sculpins, Myoxocephalus scorpius, are protected from freezing in icy seawater by alanine-rich, alpha-helical antifreeze proteins (AFPs). The major serum isoform (SS-8) has been reisolated and analyzed to establish its correct sequence. Over most of its length, this 42 amino acid protein is predicted to be an amphipathic alpha-helix with one face entirely composed of Ala residues. The other side of the helix, which is more heterogeneous and hydrophilic, contains several Lys. Computer simulations had suggested previously that these Lys residues were involved in binding of the peptide to the [11-20] plane of ice in the <-1102> direction. To test this hypothesis, a series of SS-8 variants were generated with single Ala to Lys substitutions at various points around the helix. All of the peptides retained significant alpha-helicity and remained as monomers in solution. Substitutions on the hydrophilic helix face at position 16, 19, or 22 had no obvious effect, but those on the adjacent Ala-rich surface at positions 17, 21, and 25 abolished antifreeze activity. These results, with support from our own modeling and docking studies, show that the helix interacts with the ice surface via the conserved alanine face, and lend support to the emerging idea that the interaction of fish AFPs with ice involves appreciable hydrophobic interactions. Furthermore, our modeling suggests a new N terminus cap structure, which helps to stabilize the helix, whereas the role of the lysines on the hydrophilic face may be to enhance solubility of the protein.     

Interaction among the twelve-residue segment of antifreeze protein type I,or its mutants,water and a hexagonal ice crystal

We have carried out a molecular dynamics analysis on a mixture of supercooled water, a hexagonal ice crystal and segments of winter flounder antifreeze protein. The segment consists of nine alanine residues, two threonine residues and one asparagine residue. Mutant segments, in which the threonine residues are replaced with valine residues, or serine residues, are also used. It is found that the threonine residue near the asparagine residue of the original segment is located in the vicinity of the prism face of the ice crystal. This is due to the hydrogen bond between the hydrophilic sites of these residues and water molecules, and the hydrogen bond between these water molecules and the water molecules on the ice surface. The valine and serine residues in the mutant segments do not approach the prism face of the ice crystal compared with the threonine residue near the asparagine residue. The motion of five segments, closely located side by side, is not remarkable. This is because of the gathering of water molecules caused by hydrophobic hydration, not only around alanine residues but also around the methyl sites of threonine residues.     

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.     

Structure-function relationships in an antifreeze polypeptide. The role of neutral, polar amino acids.

An alanine-rich, alpha-helical antifreeze polypeptide (AFP) from the winter flounder and seven analogs with variations in the arrangement of neutral, polar amino acids were synthesized. Circular dichroism studies determined that all of the peptides, except for one containing a proline residue, were essentially 100% alpha-helical. Freezing point depression data, analyzed by three methods, showed that rearrangement of polar residues resulted in moderate to complete loss of anti-freeze activity. It was observed that ice crystals grow as hexagonal bipyramids in dilute solutions, with a constant c to alpha axis ratio of about 3.3. Above a critical threshold concentration, which may depend on the AFP to ice binding constant and reflect the onset of cooperative interactions, growth ceases until the temperature is lowered to the freezing point. We conclude that a specific arrangement of both threonine and asparagine (or aspartic acid) residues is critical for maximal activity and that the AFPs probably bind to the pyramidal faces of ice with a specific orientation. These conclusions are consistent with a recent report (Knight, C. A., Cheng, C. C., and DeVries, A. L. (1991) Biophys. J. 59, 409-418) that a similar AFP adsorbs to the [2021] pyramidal planes of ice in dilute solution.     

Theoretical study of interaction of winter flounder antifreeze protein with ice

Antifreeze proteins (AFPs) are synthesized by various organisms to enable their cells to survive subzero environment. These proteins bind to small ice crystals and inhibit their growth, which if left uncontrolled would be fatal to cells. The crystal structures of a number of AFPs have been determined; however, crystallographic analysis of AFP-ice complex is nearly impossible. Molecular modeling studies of AFPs' interaction with ice surface is therefore invaluable. Early models of AFP-ice interaction suggested H-bond as the primary driving force behind such interaction. Recent experimental evidence, however, suggested that hydrophobic interactions could be the main contributor to AFP-ice association. All computational studies published to date were carried out to verify the H-bond model, and no works attempting to verify the hydrophobic interaction model have been published. In this work, we Monte Carlo-minimized complexes of several AFPs with ice taking into account nonbonded interactions, H-bonds, and the hydration potential for proteins. Parameters of the hydration potential for ice were developed with the assumption that the free energy of the water-ice association should be close to zero at equilibrium melting temperature. Our calculations demonstrate that desolvation of hydrophobic groups in the AFPs upon their binding to the grooves at the ice surface is indeed the major stabilizing contributor to the free energy of AFP-ice binding. This study is consistent with available structural and mutation data on AFPs. In particular, it explains the paradoxical finding that substitution of Thr residues with Val does not affect the potency of winter flounder AFP whereas substitution with Ser abolished its antifreeze activity.     

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.     

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.     

Structural variations in the alanine-rich antifreeze proteins of the pleuronectinae

The sequence and activity of antifreeze proteins from two right eye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.     

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.     

Intrahelical hydrogen bonding of serine, threonine and cysteine residues within alpha-helices and its relevance to membrane-bound proteins

A survey of known protein structures reveals that approximately 70% of serine residues and at least 85% (potentially 100%) of threonine residues in helices make hydrogen bonds to carbonyl oxygen atoms in the preceding turn of the helix. The high frequency of intrahelical hydrogen bonding is of particular significance for intrinsic membrane-bound proteins that form transmembrane helices. Hydrogen bonding within a helix provides a way for serine, threonine and cysteine residues to satisfy their hydrogen-bonding potential permitting such residues to occur in helices buried within a hydrophobic milieu.     

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]     

A theoretical model of a plant antifreeze protein from Lolium perenne.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein's inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.