Ice-binding site of surface-bound type III antifreeze protein partially decoupled from water.

Type III antifreeze proteins (AFP III) have been widely recognized as one class of ice-binding proteins produced by several biological organisms to withstand freezing conditions. Besides their ability to restrict ice growth through their ice-binding site (IBS), AFP III have also been shown to possess a great propensity for hydrophobic surfaces such as the air-water interface. Yet, it is not known whether AFP III adsorb with a specific orientation and how hydrophobic interactions affect the IBS. Molecular insights on the accessibility of the IBS and its interactions with water are important for understanding AFP III action in vivo but also for their application as ice-inhibiting agents for deicing, frozen food storage, as well as for long-term blood and organ cryo-preservation. Here, the orientation of fish AFP III adsorbed at the air-water interface has been studied using a combination of molecular dynamics (MD) simulations and vibrational sum-frequency generation (SFG) spectroscopy together with spectral calculations. The SFG/MD analysis indicated that when AFP III adsorbs at the air-water interface, it mostly retains its native state and orients with a tilt angle of 120° with respect to the surface normal. We found that the IBS is only partially solvated, leaving the pyramidal ice plane binding domain exposed to the vapor phase. These findings suggest that interactions with hydrophobic interfaces (e.g., cell membranes, polymers) could lead to the partial decoupling of the IBS from water and, to some extent, to a loss of AFP III antifreezing activity.

NMR study of the antifreeze activities of active and inactive isoforms of a type III antifreeze protein.

The quaternary-amino-ethyl 1 (QAE1) isoforms of type III antifreeze proteins (AFPs) prevent the growth of ice crystals within organisms living in polar regions. We determined the antifreeze activity of wild-type and mutant constructs of the Japanese notched-fin eelpout (Zoarces elongates Kner) AFP8 (nfeAFP8) and characterized the structural and dynamics properties of their ice-binding surface using NMR. We found that the three constructs containing the V20G mutation were incapable of stopping the growth of ice crystals and exhibited structural changes, as well as increased conformational flexibility, in the first 310 helix (residues 18-22) of the sequence. Our results suggest that the inactive nfeAFP8s are incapable of anchoring water molecules due to the unusual and flexible backbone conformation of their primary prism plane-binding surface.

Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity.

Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the mutations. Therefore, we do not observe any correlation between the antifreeze activity and the extent of modification of the properties of solvation water.

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two ß-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

The protective role of antifreeze protein 3 on the structure and function of mature mouse oocytes in vitrification.

Several studies have reported the oocyte damage in mice during vitrification; however, little has been known about the protective role that antifreeze protein 3 (Afp3) plays on their cellular structure and function during vitrification. In order to observe the extracellular cryo-protective role of Afp3, four groups were divided randomly. The observations were made for changes in cytoskeleton, expression of the related genes before and after vitrification, and also for changes in the in vitro developmental potential of oocytes. The outcomes were as follows: (i) microtubules, actin filaments and chromosomal integrity were more intact in the vitrification group supplemented with additional Afp3 compared to the vitrification group. In the fresh control group and the group with additional cryoprotectant containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) and sucrose, the organelles were more intact than the other two vitrification groups. (ii) Real-time PCR analysis revealed that the relative quantification of mitotic arrest deficient 2 (Mad2) and centromere protein E (Cenp-e) were significantly higher in the vitrification group with additional Afp3, the fresh control group and the one group with additional cryoprotectant, in comparison to the vitrification group. On the contrary, the expression of cold inducible RNA-binding protein (Cirbp) and kinesin-5 motor protein (Eg5) were up-regulated in the vitrification group compared to the remaining groups. (iii) The fertilization rate and the recovery rate in the fresh control group and the group with additional cryoprotectant were higher than the other two vitrification groups; furthermore, the recovery rate and the fertilization rate in the vitrification group with Afp3 were higher than the vitrification group. However, the blastocyst formation rate in all the four groups showed no statistical significance. In conclusion, Afp3 plays a positive role in the structure and function of mice oocytes in vitrification.

Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus.

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice.

Type III antifreeze proteins (AFPs) can be sub-divided into three classes of isoforms. SP and QAE2 isoforms can slow, but not stop, the growth of ice crystals by binding to pyramidal ice planes. The other class (QAE1) binds both pyramidal and primary prism planes and is able to halt the growth of ice. Here we describe the conversion of a QAE2 isoform into a fully-active QAE1-like isoform by changing four surface-exposed residues to develop a primary prism plane binding site. Molecular dynamics analyses suggest that the basis for gain in antifreeze activity is the formation of ice-like waters on the mutated protein surface.

Protein-ice interaction of an antifreeze protein observed with solid-state NMR.

NMR on frozen solutions is an ideal method to study fundamental questions of macromolecular hydration, because the hydration shell of many biomolecules does not freeze together with bulk solvent. In the present study, we present previously undescribed NMR methods to study the interactions of proteins with their hydration shell and the ice lattice in frozen solution. We applied these methods to compare solvent interaction of an ice-binding type III antifreeze protein (AFP III) and ubiquitin a non-ice-binding protein in frozen solution. We measured (1)H-(1)H cross-saturation and cross-relaxation to provide evidence for a molecular contact surface between ice and AFP III at moderate freezing temperatures of -35 °C. This phenomenon is potentially unique for AFPs because ubiquitin shows no such cross relaxation or cross saturation with ice. On the other hand, we detected liquid hydration water and strong water-AFP III and water-ubiquitin cross peaks in frozen solution using relaxation filtered (2)H and HETCOR spectra with additional (1)H-(1)H mixing. These results are consistent with the idea that ubiquitin is surrounded by a hydration shell, which separates it from the bulk ice. For AFP III, the water cross peaks indicate that only a portion of its hydration shell (i.e., at the ice-binding surface) is in contact with the ice lattice. The rest of AFP III's hydration shell behaves similarly to the hydration shell of non-ice-interacting proteins such as ubiquitin and does not freeze together with the bulk water.

Structure and interactions of fish type III antifreeze protein in solution.

It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL(-1) and at temperatures of 20 degrees C, 6 degrees C, and 4 degrees C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.

Fully active QAE isoform confers thermal hysteresis activity on a defective SP isoform of type III antifreeze protein.

Type III antifreeze protein is naturally expressed as a mixture of sulfopropyl-Sephadex (SP) and quaternary aminoethyl-Sephadex (QAE)-binding isoforms, whose sequence identity is approximately 55%. We studied the ice-binding properties of a SP isoform (nfeAFP6) and the differences from those of a QAE isoform (nfeAFP8); both of these isoforms have been identified from the Japanese fish Zoarces elongatus Kner. The two isoforms possessed ice-shaping ability, such as the creation of an ice bipyramid, but nfeAFP6 was unable to halt crystal growth and exhibited no thermal hysteresis activity. For example, the ice growth rate for nfeAFP6 was 1000-fold higher than that for nfeAFP8 when measured for 0.1 mm protein solution at 0.25 degrees C below the melting point. Nevertheless, nfeAFP6 exhibited full thermal hysteresis activity in the presence of only 1% nfeAFP8 (i.e. [nfeAFP8]/[nfeAFP6] = 0.01), the effectiveness of which was indistinguishable from that of nfeAFP8 alone. We also observed a burst of ice crystal growth from the tip of the ice bipyramid for both isoforms on lowering the temperature. These results suggest that the ice growth inhibitory activity of an antifreeze protein isoform lacking the active component is restored by the addition of a minute amount of the active isoform.

Modified Langmuir isotherm for a two-domain adsorbate: derivation and application to antifreeze proteins.

Many organisms are exposed to subzero temperatures in nature and can survive these temperatures by the effect of antifreeze proteins (AFPs), which inhibit ice crystal growth and change the morphology of ice crystals. Although the effects of these proteins, such as recrystallization inhibition, ice growth inhibition, and crystal habit changes, are known, a conclusive description of the protein-ice crystal interaction including interaction energy, surface coverage, and lifetime of adsorbate has been elusive. In this study, we determine the binding equilibrium constant for a type III fish antifreeze protein and the relationship between thermal hysteresis and surface coverage for this protein. This is possible using experimental data from a two-domain antifreeze protein and its related single domain protein. The classical Langmuir isotherm is used to describe the equilibrium exchange of the single domain type III AFP molecules at the ice crystal surface, while a modification of the Langmuir isotherm is derived to describe the adsorption of the two-domain AFP. Because the protein adsorption is governed by different isotherm relationships, there are two independent data sets allowing the determination of the two unknowns of surface coverage and binding energy. The data yield an equilibrium binding constant of 1.9 mM(-1) for the type III AFP-ice interaction. The analysis results in a relationship between surface coverage and thermal hysteresis, as well as kinetic equations of the adsorption of the proteins onto the ice surface.

Solid-state NMR on a type III antifreeze protein in the presence of ice.

Antifreeze proteins (AFPs) are found in fish, insects, plants, and a variety of other organisms where they serve to prevent the growth of ice at subzero temperatures. Type III AFPs cloned from polar fishes have been studied extensively with X-ray crystallography, liquid-state NMR, and site directed mutagenesis and are, therefore, among the best characterized AFPs. A flat surface on the protein has previously been proposed to be the ice-binding site of type III AFP. The detailed nature of the ice binding remains controversial since it is not clear whether only polar or also hydrophobic residues are involved in ice binding and there is no structural information available of a type III AFP bound to ice. Here we present a high-resolution solid-state NMR study of a type III AFP (HPLC-12 isoform) in the presence of ice. The chemical-shift differences we detected between the frozen and the nonfrozen state agree well with the proposed ice-binding site. Furthermore, we found that the (1)H T(1) of HPLC-12 in frozen solution is very long compared to typical (1)H of proteins in the solid state as for example of ubiquitin in frozen solution.

Hypothermic preservation effect on mammalian cells of type III antifreeze proteins from notched-fin eelpout.

Antifreeze proteins (AFPs) can bind to the surface of ice crystals and have also been suggested to protect cells from hypothermic damage. The present study reports that type III AFPs from notched-fin eelpout, Zoarces elongatus Kner, can protect cells during hypothermic storage. This fish naturally expresses at least 13 isoforms of type III AFP (denoted NfeAFPs), the primary sequences of which were categorized into SP- and QAE-Sephadex binding groups (SP- and QAE-isoforms). We compared the preservation ability between the extracted isoform mixtures (NfeAFPs) and a recombinant single SP-isoform (RcNfeAFP6). Experiments were performed using cultivated mammalian cells (HepG2) exposed to 4 degrees C for 24-72 h. The preserved cells were evaluated by measuring LDH released, intracellular ATP, and WST-8 reduction. It appeared that the protective effect of the 2 samples increases dose-dependently at concentrations between 2 and 10 mg/ml. Under highest soluble amount of the protein (approximately 10 mg/ml), cell viability significantly improved compared with the ordinary preservation fluid (P<0.01). This effect was larger with NfeAFPs than with RcNfeAFP6 at the same concentration. The successful hypothermic preservation of cells using natural NfeAFPs may have a wide range of applications for cell engineering and clinical medical care.

Two domains of RD3 antifreeze protein diffuse independently.

Antifreeze proteins (AFPs) make up a class of structurally diverse proteins that help to protect many organisms from freezing temperatures by inhibiting ice crystal growth at temperatures below the colligative freezing point. AFPs are typically small proteins with a relatively flat, slightly hydrophobic binding region that matches the lattice structure of a specific ice crystal plane. The only known two-domain AFP is RD3 from the Antarctic eel pout. It consists of two nearly identical type III domains connected by a nine-residue linker. This protein exhibits higher activity than the single-domain protein at low concentrations. The initial solution structure of RD3 revealed that the domains were aligned so that the binding regions were nearly coplanar, effectively doubling the surface area for binding. A more recent report suggests that the domains may not be aligned in solution but rather diffuse independently. To resolve the issue, we have measured the NMR residual dipolar couplings using alignment media of stretched gels and filamentous phage to determine the relative orientation of the domains. We find that the two domains of RD3 are free to move relative to each other, within the constraint of the flexible nine-residue linker. Our data show that there is no strongly preferred alignment in solution. Furthermore, the flexibility and length of the linker are sufficient to allow the two domains to have their binding faces in the same orientation and coplanar for simultaneous binding to an ice crystal surface.

Temperature-induced unfolding pathway of a type III antifreeze protein: insight from molecular dynamics simulation.

Molecular dynamics simulations of the temperature-induced unfolding reaction of a cold-adapted type III antifreeze protein (AFPIII) from the Antarctic eelpout Lycodichthys dearborni have been carried out for 10 ns each at five different temperatures. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. Plots of backbone root mean square deviation (RMSD) against radius of gyration (Rg) serve as phase space trajectories. These plots also indicate that the protein unfolds without many detectable intermediates suggestive of two-state unfolding kinetics. The transition state structures are identified from essential dynamics, which utilizes a principal component analysis (PCA) on the atomic fluctuations throughout the simulation. Overall, the transition state resembles an expanded native state with the loss of the three 3(10) helices and disrupted C-terminal region. Our study provides insight into the structure-stability relationship of AFPIII, which may help to engineer AFPs with increased thermal stability that is more desirable than natural AFPs for some industrial and biomedical purposes.

Determination of the expression of fish antifreeze protein (AFP) in 7th generation transgenic mice tissues and serum.

In this study, the presence of antifreeze protein (AFP) gene expression through successive generations in transgenic mice carrying the chimeric gene construct of the coding sequence for the AFP protein from ocean pout was investigated. AFP transgenic hemizygote mice were used for AFP gene expression. AFP genome expressions in transgenic mice were analyzed by Western blotting, and tissue location of AFP protein was shown by immunohistochemical and immunofluorescence techniques. Seventh transgenic mice from the established founders demonstrated the expression of AFP in organs such as the skin, oviduct, lung, kidney and liver tissues and serum except for the heart. Our results demonstrate successful expression of AFP gene products in several tissues and serum of transgenic mice, the association of in vivo expressed AFP protein, for the first time. These results indicate that the coding sequence for the AFP protein gene (ocean pout type III AFP gene) could be integrated and stably transcribed and expressed in the 7th generation of transgenic mice. In conclusion transgenic mouse lines would be a good model for the cryostudy of AFP and for the determination of AFP roles in several organs and tissues.

Tissue specific expression of antifreeze protein and growth hormone transgenes driven by the ocean pout (Macrozoarces americanus) antifreeze protein OP5a gene promoter in Atlantic salmon (Salmo salar).

Previous research aimed at producing genetically improved salmon broodstock for aquaculture led to the creation of two lines of transgenic Atlantic salmon using gene constructs that were derived in part from the ocean pout OP5a antifreeze protein (AFP) gene. One of the lines was produced using an OP5a AFP gene in which the 5' region of the promoter was removed (termed t-OP5a-AFP), and the other line contains a growth hormone (GH) transgene (EO-1alpha) that consists of a chinook salmon GH cDNA driven by a truncated OP5a AFP promoter that is almost identical to that of the t-OP5a-AFP construct. The similarity of the promoter regions of these transgenes provided an opportunity to evaluate their tissue specific expression patterns. Expression of mRNA was evaluated using Northern blot and RT-PCR techniques. The results demonstrate that the AFP and GH trangenes were expressed in almost all body tissues, suggesting that the promoter region of the OP5a AFP gene lacks tissue specific elements. Northern analysis revealed that expression of the t-OP5a-AFP gene was considerably greater than that of the EO-1alpha GH transgene. Only the spleen tissue of the GH transgenics showed a visible band of hybridization. In contrast clear bands of hybridization were evident in all tissues, except for blood cells, of the AFP transgenics with heart, liver and brain tissue showing the highest levels of mRNA expression. This higher level of expression could be attributable to the presence of introns in the t-OP5a-AFP transgene. Since the GH transgenic salmon grow considerably faster than non-transgenics the low levels of GH transgene expression in this line were clearly sufficient to produce the desired rapid growth phenotype. In contrast the levels of AFP expression were inadequate to impart any improvement in the freeze resistance of the AFP transgenic salmon.

Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges.

A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein (AFP), was carried out. The results indicate that the folding equilibrium of type III AFP is a reversible, unimolecular, two-state process with no populated intermediates. Compared to most mesophilic proteins whose folding is two-state, the psychrophilic type III AFP has a much lower thermodynamic stability at 25 degrees C, approximately 3 kcal/mol, and presents a remarkably downshifted stability-temperature curve, reaching a maximum of 5 kcal/mol around 0 degrees C. Type III AFPs contain few and non-optimally distributed surface charges relative to their mesophilic homologs, the C-terminal domains of sialic acid synthases. We used thermodynamic double mutant cycles to evaluate the energetic role of every surface salt bridge in type III AFP. Two isolated salt bridges provided no contribution to stability, while the Asp36-Arg39 salt bridge, involved in a salt bridge network with the C-terminal carboxylate, had a substantial contribution (approximately 1 kcal/mol). However, this contribution was more than counteracted by the destabilizing effect of the Asp36 carboxylate itself, whose removal led to a net 30% increase in stability at 25 degrees C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0 degrees C) at which AFPs must function. In addition, it indicates that salt bridge networks are used in nature also for the stability of psychrophilic proteins, and has led to a type III AFP variant of increased stability that could be used for biotechnological purposes.

The mechanism of the type III antifreeze protein action: a computational study.

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.