Ice-binding Surface Research Articles

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.

Computational Study on the Function of Water within a β-Helix Antifreeze Protein Dimer and in the Process of Ice-Protein Binding

Antifreeze proteins (AFPs) help many organisms protect themselves from freezing in subzero temperatures. The most active AFPs found to date are those from insects, which possess exceptionally regular β-helical structures. On the ice-binding surface of these proteins, regularly arrayed water molecules are observed within the repeating Thr-Xxx-Thr motif, but the exact role of these water molecules remains unknown. In this work, we have employed a number of computational methods to examine the role of these water molecules in an AFP from Tenebrio molitor (TmAFP). Our investigation involved a combination of molecular and quantum mechanical approaches. Properties such as stability, interaction energy, orbital overlap, and conformational analysis of various systems, including TmAFP-water, TmAFP-water-ice, and TmAFP-ice, were systematically evaluated and compared. The regularly arrayed water molecules were found to remain associated with TmAFP before ice binding, demonstrating that they are an intrinsic part of the protein. These water molecules may assist TmAFP in the process of ice recognition and binding. However, after facilitating the initial stages of ice recognition and binding, these water molecules are excluded in the final formation of the AFP-ice complex. The departure of these water molecules enables a better two-dimensional match between TmAFP and ice. These results agree with experimental observations showing that although these water molecules are aligned with the ice-binding hydroxyl groups of Thr residues in one dimension, they are in fact positioned slightly off in the second dimension, making a good two-dimensional match impossible.

Analysis of thermal hysteresis protein hydration using the random network model

The hydration of polar and apolar groups can be explained quantitatively, via the random network model of water, in terms of differential distortions in first hydration shell water–water hydrogen bonding angle. This method of analyzing solute induced structural distortions of water is applied to study the ice-binding type III thermal hysteresis protein. The analysis reveals subtle but significant differences in solvent structuring of the ice-binding surface, compared to non-ice binding protein surface. The major differences in hydration in the ice-binding region are (i) polar groups have a very apolar-like hydration. (ii) there is more uniform hydration structure. Overall, this surface strongly enhances the tetrahedral, or ice-like, hydration within the primary hydration shell. It is concluded that these two specific features of the hydration structure are important for this surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.

The Refined Crystal Structure of an Eel Pout Type III Antifreeze Protein RD1 at 0.62-Å Resolution Reveals Structural Microheterogeneity of Protein and Solvation

RD1 is a 7-kDa globular protein from the Antarctic eel pout Lycodichthys dearborni. It belongs to type III of the four types of antifreeze proteins (AFPs) found in marine fishes living at subzero temperatures. For type III AFP, a potential ice-binding flat surface has been identified and is imbedded with side chains capable of making hydrogen bonds with a specific lattice plane on ice. So far, all crystallographic studies on type III AFPs were carried out using the Atlantic ocean pout Macrozoarces americanus as the source organism. Here we present the crystal structure of a type III AFP from a different zoarcid fish, and at an ultra-high resolution of 0.62 Å. The protein fold of RD1 comprises a compact globular domain with two internal tandem motifs arranged about a pseudo-dyad symmetry. Each motif of the “pretzel fold” includes four short β-strands and a 3 10 helix. There is a novel internal cavity of 45 Å 3 surrounded by eight conserved nonpolar residues. The model contains several residues with alternate conformations, and a number of split water molecules, probably caused by alternate interactions with the protein molecule. After extensive refinement that includes hydrogen atoms, significant residual electron densities associated with the electrons of peptides and many other bonds could be visualized.

Reversible binding of the HPLC6 isoform of type I antifreeze proteins to ice surfaces and the antifreeze mechanism studied by multiple quantum filtering-spin exchange NMR experiment.

Antifreeze proteins (AFPs) protect organisms from freezing damage by inhibiting the growth of seed-ice crystals. It has long been hypothesized that irreversible binding of AFPs to ice surfaces is responsible for inhibiting the growth of seed-ice crystals as such a mechanism supports the popularly accepted Kelvin effect for the explanation of local freezing-point depression. However, whether the binding is reversible or irreversible is still under debate due to the lack of direct experimental evidence. Here, we report the first direct experimental result, by using the newly developed multiple quantum (MQ) filtering-spin exchange NMR experiment, that shows that the binding of HPLC6 peptides to ice surfaces is reversible. It was found that the reversible process can be explained by the model of monolayer adsorption. These results suggest that the Kelvin effect is not suitable for explaining the antifreeze mechanism, and direct interactions between the peptides and the ice-surface binding sites are the driving forces for the binding of AFPs to ice surfaces. We propose that there exists a concentration gradient of AFP from an ice-binding surface to the solution due to the affinity of ice surfaces to AFPs. This concentration gradient creates a dense layer of AFP in contact with the ice-binding surface, which depresses the local freezing point because of the colligative property, but not the Kelvin effect.

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.

A β-Helical Antifreeze Protein Isoform with Increased Activity: STRUCTURAL AND FUNCTIONAL INSIGHTS

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular beta-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Delta-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its beta-helical structure and hence the size of its ice-binding face.

Positive darwinian selection promotes heterogeneity among members of the antifreeze protein multigene family.

A variety of organisms have independently evolved proteins exhibiting antifreeze activity that allows survival at subfreezing temperatures. The antifreeze proteins (AFPs) bind ice nuclei and depress the freezing point by a noncolligative absorption-inhibition mechanism. Many organisms have a heterogeneous suite of AFPs with variation in primary sequence between paralogous loci. Here, we demonstrate that the diversification of the AFP paralogues is promoted by positive Darwinian selection in two independently evolved AFPs from fish and beetle. First, we demonstrate an elevated rate of nonsynonymous substitutions compared to synonymous substitutions in the mature protein coding region. Second, we perform phylogeny-based tests of selection to demonstrate a subset of codons is subjected to positive selection. When mapped onto the three-dimensional structure of the fish antifreeze type III antifreeze structure, these codons correspond to amino acid positions that surround but do not interrupt the putative ice-binding surface. The selective agent may be related to efficient binding to diverse ice surfaces or some other aspect of AFP function.

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.

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 β-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.

Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein.

Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-A resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat beta-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section.

Ice-Binding Surface of Fish Type III Antifreeze

We employed computational techniques, including molecular docking, energy minimization, and molecular dynamics simulation, to investigate the ice-binding surface of fish type III antifreeze protein (AFP). The putative ice-binding site was previously identified by mutagenesis, structural analysis, and flatness evaluation. Using a high-resolution x-ray structure of fish type III AFP as a model, we calculated the ice-binding interaction energy of 11 surface patches chosen to cover the entire surface of the protein. These various surface patches exhibit small but significantly different ice-binding interaction energies. For both the prism ice plane and an “ice” plane in which water O atoms are randomly positioned, our calculations show that a surface patch containing 14 residues (L19, V20, T18, S42, V41, Q9, P12, A16, M21, T15, Q44, I13, N14, K61) has the most favorable interaction energy and corresponds to the previously identified ice-binding site of type III AFP. Although in general agreement with the earlier studies, our results also suggest that the ice-binding site may be larger than the previously identified “core” cluster that includes mostly hydrophilic residues. The enlargement mainly results from the inclusion of peripheral hydrophobic residues and K61.

A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor.

We have previously identified a Thr- and Cys-rich thermal hysteresis (antifreeze) protein (THP) in the beetle Tenebrio molitor that has 10-100 times the freezing point depression activity of fish antifreeze proteins. Because this 8.4 kDa protein is significantly different in its properties from THP preparations previously reported from this insect, a thorough search was undertaken for other antifreeze types. Many active proteins were observed, but all appeared to be isoforms of the THP that differed in their number of 12-amino acid repeats (consensus sequence CTxSxxCxxAxT), amino acid substitutions, and N-linked glycosylation. Mass spectral analysis has matched most of these isoforms with cDNA sequences of 17 different clones from a larval fat body library that encode eight different mature THPs containing 84, 96, or 120 amino acids. Genomic Southern blots suggest there may be 30-50 tightly linked copies of the gene, which is a signature consistently seen with unrelated fish antifreeze protein genes, and one that has been associated with the need to rapidly increase gene product in response to climate change. A three-dimensional model is proposed for the fully disulfide-bonded structure of T. molitor THP, which can accommodate addition or deletion of 12-amino acid repeats. The structure is a beta-helix that places most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.

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.

Identification of the Ice-Binding Surface on a Type III Antifreeze Protein with a “Flatness Function” Algorithm

Antifreeze proteins (AFPs) adsorb to surfaces of growing ice crystals, thereby arresting their growth. The prevailing hypothesis explains the nature of adsorption in terms of a match between the hydrophilic side chains on the AFP's ice-binding surface (IBS) and the water molecules on the ice surface. The number and spatial arrangement of hydrogen bonds thus formed have been proposed to account, respectively, for the binding affinity and specificity. The crystal structure of a type III AFP from ocean pout (isoform HPLC-3) has been determined to 2.0-A resolution. The structure reveals an internal dyad motif formed by two 19-residue, loop-shaped elements. Based on of the flatness observed on the type I alpha-helical AFP's IBS, an automated algorithm was developed to analyze the surface planarity of the globular type III AFP and was used to identify the IBS on this protein. The surface with the highest flatness score is formed by one loop of the dyad motif and is identical to the IBS deduced from earlier mutagenesis studies. Interestingly, 67% of this surface contains nonpolar solvent-accessible surface area. The success of our approach to identifying the IBS on an AFP, without considering the presence of polar side chains, indicates that flatness is the first approximation of an IBS. We further propose that the specificity of interactions between an IBS and a particular ice-crystallographic plane arises from surface complementarity.

The effects of steric mutations on the structure of type III antifreeze protein and its interaction with ice

The interaction of proteins with ice is poorly understood and difficult to study, partly because ice is transitory and can present many binding surfaces, and partly because structures have been determined for only two ice-binding proteins. This paper focuses on one of these, a 66-residue antifreeze protein (AFP) from eel pout. The high resolution X-ray structure of this fish AFP demonstrated that the proposed ice-binding surface is remarkably flat for such a small protein. The residues on the planar surface thought to be involved in ice binding are restrained by hydrogen bonds or by tight packing of their side-chains. To probe the requirement for a flat binding surface, a conserved alanine in the center of the AFP planar surface was substituted with larger residues. Six alanine replacement mutants (Ala16>Cys, Thr, Met, Arg, His and Tyr), designed to disrupt the planarity of the surface and sterically block binding to ice, were characterized by X-ray crystallography and compared with the wild-type AFP. In each case, the detail provided by these crystal structures has helped explain the effects of the mutation on antifreeze activity. The substitutions, Ala16>His and Ala16>Tyr, were large enough to shield Gln44, one of the putative ice-binding residues, contributing to their very low thermal hysteresis activity. In addition to sterically hindering the putative ice-binding site, the bulkier residues also caused shifts in the putative ice-binding residues owing to the tight packing of side-chains on the planar surface. This unexpected consequence of the mutations helps account for the severely reduced antifreeze activity. One explanation for residual antifreeze activity in some of the mutants lies in the possibility that AFPs have a role in shaping the site on the ice to which they bind. Thus, side-chain dislocations might be partially accommodated by ice that can freeze around them. It is evident that the disruption of the planarity, by introducing larger residues at the center of the proposed ice-binding site, is not the only factor responsible for the loss of antifreeze activity. There are multiple causes including positional change and steric blockage of some putative ice-binding residues.

Structure‐function relationship in the globular type III antifreeze protein: Identification of a cluster of surface residues required for binding to ice

Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 beta-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal beta-sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154-1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal beta-sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP-ice interaction.