Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.     

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.     

Expression of a cystine-rich fish antifreeze in transgenicDrosophila melanogaster

We have usedDrosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting.Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 °C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenicDrosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.     

Activity of short segments of Type I antifreeze protein

In this work, we present a study on the antifreeze activity of short segments of a Type I antifreeze protein, instead of the whole protein. This approach simplifies the correlation between antifreeze protein characteristics, such as hydrophilicity/hydrophobicity, and the effect of these characteristics on the antifreeze mechanism. Three short polypeptides of Type I AFP have been synthesized. Their antifreeze activity and interactions with water and ice crystals have been analyzed by various techniques such as circular dichroism spectroscopy, X-ray diffraction, differential scanning calorimetry, and osmometry. It is shown that one short segment of Type I AFP has an antifreeze activity of about 60% of the native protein activity. In this work, we demonstrate that short segments of Type I AFPs possess nonzero thermal hysteresis and result in modifications in the growth habits and growth rates of ice. This approach enables the preparation of large quantities of short AFP segments at low cost with high antifreeze activity, and opens the possibility of developing the commercial potential of AFPs.     

The role of N and C termini in the antifreeze activity of winter flounder (Pleuronectes americanus) antifreeze proteins

Antifreeze proteins (AFPs) are found in many marine fish and have been classified into five biochemical classes: AFP types I-IV and the antifreeze glycoproteins. Type I AFPs are alpha-helical, partially amphipathic, Ala-rich polypeptides. The winter flounder (Pleuronectes americanus) produces two type I AFP subclasses, the liver-type AFPs (wflAFPs) and the skin-type AFPs (wfsAFPs), that are encoded by distinct gene families with different tissue-specific expression. wfsAFPs and wflAFPs share a high level of identity even though the wfsAFPs have approximately half the activity of the wflAFPs. Synthetic polypeptides based on two representative wflAFPs and wfsAFPs were generated to examine the role of the termini in antifreeze activity. Through systematic exchange of N and C termini between wflAFP-6 and wfsAFP-2, the termini were determined to be the major causative agents for the variation in activity levels between the two AFPs. Furthermore, the termini of wflAFP-6 possessed greater helix-stabilizing ability compared with their wfsAFP-2 counterparts. The observed 50% difference in activity between wflAFP-6 and wfsAFP-2 can be divided into approximately 20% for differences at each termini and approximately 10% for differences in the core. Furthermore, the N terminus was determined to be the most critical component for antifreeze activity.     

Functional Diversification and Evolution of Antifreeze Proteins in the Antarctic Fish Lycodichthys dearborni

Antifreeze proteins (AFPs) have independently evolved in many organisms. AFPs act by binding to ice crystals, effectively lowering the freezing point. AFPs are often at high copy number in a genome and diversity exists between copies. Type III antifreeze proteins are found in Arctic and Antarctic eel pouts, and have previously been shown to evolve under positive selection. Here we combine molecular and proteomic techniques to understand the molecular evolution and diversity of Type III antifreeze proteins in a single individual Antarctic fish Lycodichthys dearborni. Our expressed sequence tag (EST) screen reveals that at least seven different AFP variants are transcribed, which are ultimately translated into five different protein isoforms. The isoforms have identical 66 base pair signal sequences and different numbers of subsequent ice-binding domains followed by a stop codon. Isoforms with one ice-binding unit (monomer), two units (dimer), and multiple units (multimer) were present in the EST library. We identify a previously uncharacterized protein dimer, providing further evidence that there is diversity between Type III AFP isoforms, perhaps driven by positive selection for greater thermal hysteresis. Proteomic analysis confirms that several of these isoforms are translated and present in the liver. Our molecular evolution study shows that paralogs have diverged under positive selection. We hypothesize that antifreeze protein diversity is an important contributor to depressing the serum freezing point.     

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.     

Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.     

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. Received: 27 February 2001 / Accepted: 12 September 2001     

昆虫抗冻蛋白基因的克隆与表达研究进展

产生抗冻蛋白(antifreeze protein,AFP)是许多昆虫抵御寒冷的一种重要机制。昆虫抗冻蛋白基因的克隆和表达是研究抗冻蛋白活性和功能的主要途径。文章归纳GenBank所登录的昆虫抗冻蛋白基因及其特点,总结昆虫抗冻蛋白基因的天然表达和基因工程表达方面尚未明确或需要克服的一些问题。目前在GenBank注册的昆虫抗冻蛋白基因约100个,集中于9种昆虫隶属鞘翅目3个科和鳞翅目1个科。昆虫抗冻蛋白基因具有多拷贝和多同种型(isoforms)的特点。昆虫抗冻蛋白的天然表达具有物种间和同种型间的多样性。基因工程表达昆虫抗冻蛋白需要克服表达量低活性不高的问题。对昆虫抗冻蛋白表达规律的研究有助于全面认识其功能。     

Effect of glycosylation on hydration behavior at the ice-binding surface of the Ocean Pout type III antifreeze protein: a molecular dynamics simulation

Antifreeze proteins (AFPs), found in certain vertebrates, plants, fungi and bacteria have the ability to permit their survival in subzero environments by thermal hysteresis mechanism. However, the exact mechanism of ice growth inhibition is still not clearly understood. Here, four long explicit molecular dynamics (MD) simulations have been carried out at two different temperatures (277 and 298 K) with and without glycan to study the conformational rigidity of the Ocean pout type III antifreeze protein in aqueous medium and the structural arrangements of water molecules hydrating its ice-binding surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its non ice-binding surface (NonIBS) in its native and glycosylated form. Hydrophilic residues N14, T18 and Q44 are essential to antifreeze activity. Radial distribution, density distribution function and nearest neighbor orientation plots with respect to individual two surfaces confirm that density of water molecule near these binding surface in native and glycosylated form are relatively more than the nonbinding surface. The glycosylated form shows a strong peak than the native one. From rotational auto correlation function of water molecules around ice-binding sites, it is prominent that with increase in temperature, strong interaction between the water oxygen and the hydrogen bond acceptor group on the protein-binding surface decreases. This provides a possible molecular reason behind the ice-binding activity of ocean pout at the prism plane of ice.     

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.     

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.     

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.     

Antifreeze proteins in higher plants

Overwintering plants produce antifreeze proteins (AFPs) having the ability to adsorb onto the surface of ice crystals and modify their growth. Recently, several AFPs have been isolated and characterized and five full-length AFP cDNAs have been cloned and characterized in higher plants. The derived amino acid sequences have shown low homology for identical residues. Theoretical and experimental models for structure of Lolium perenne AFP have been proposed. In addition, it was found that the hormone ethylene is involved in regulating antifreeze activity in response to cold. In this review, it is seen that the physiological and biochemical roles of AFPs may be important to protect the plant tissues from mechanical stress caused by ice formation.     

差示扫描量热法直接测定沙冬青抗冻蛋白的热滞效应

用差示扫描量热法直接测定了从沙冬青中提取的一种抗冻蛋白（ＡＦＰ）组分的低温热行为。结果表明,该组分的低温热行为远较文献报道的各种抗冻蛋白复杂。在降、升温过程中,在低和高温侧都给出两个放或吸热峰,两个峰表现出相互独立而又相互依存的热滞行为。低温峰的热滞活性远高于高温岭。我们认为,这种ＡＦＰ分子对水及冰晶很可能有两种不同的相互作用和影响。     

昆虫对低温的适应——抗冻蛋白研究进展

昆虫抗冻蛋白的研究主要在几种昆虫中展开,到目前为止已有二十多种昆虫抗冻蛋白被分离纯化。本文综述了关于昆虫抗冻蛋白的结构、组成、生物学活性及功能等方面的研究进展。昆虫抗冻蛋白的二级结构为β折叠和β转角,在其特殊的氨基酸序列结构中,半胱氨酸形成的二硫键对稳定其结构和活性起着很重要的作用。影响昆虫抗冻蛋白的因子,如活化蛋白及低分子量溶质的发现开辟了昆虫抗冻蛋白研究的新领域。     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9 °C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.     

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.