Biosynthesis of winter flounder antifreeze proprotein in E.coli

A semisynthetic winter flounder antifreeze proprotein (proAFP) coding region was constructed and inserted into a lacZ expression vector. ProAFP was produced from the vector in Escherichia coli as a C-terminal fusion to the first 289 amino acids of beta-galactosidase (beta-gal). The proAFP and beta-gal domains of the beta-gal-proAFP fusion protein were separated by the recognition signal for the blood coagulation protease, factor Xa. Upon induction with isopropylthio-beta-D-galactoside the fusion protein accumulated to levels of 15% of the total protein. The beta-gal-proAFP fusion protein was partially purified by differential centrifugation, but required solubilization prior to factor Xa digestion. The solubilized fusion protein was efficiently and correctly cleaved by factor Xa, after which the proAFP was purified by gel permeation. Bacterial proAFP was indistinguishable from natural proAFP by the criteria of antifreeze activity, amino-terminal sequence (15 cycles), reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis. Circular dichroism measurements showed that proAFP is a composite of random coil and alpha-helical secondary structure, with an alpha-helix content of 44% at 0 degrees C. It seems probable that the C-terminal region of proAFP, which corresponds to the mature AFP protein, is mainly alpha-helical, and that the N-terminal pro-segment is random coiled.     

Secretory expression and site-directed mutagenesis studies of the winter flounder skin-type antifreeze polypeptides.

Winter flounder contains both liver-type, extracellular antifreeze polypeptides (wflAFPs) and less active skin-type, intracellular antifreeze polypeptides (wfsAFPs). The lower activity of wfsAFPs might be due to their lack of complete ice-binding motifs '-K-DT-'. In order to test the functional role of this putative ice-binding motif, mutations were introduced into the N-terminal or C-terminal regions of wfsAFP-2, which lack any presumptive ice-binding motifs. The wild-type and mutant wfsAFP-2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity. Surprisingly, the antifreeze activity decreased with the introduction of ice-binding motifs. However, there was a corresponding decrease in alpha-helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice-binding motifs. These studies have brought new definitions of the roles of ice-binding motif residues in type I antifreeze proteins.     

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

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

Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder antifreeze protein gene

We have analyzed the inheritance and expression of a line of transgenic salmon harboring the antifreeze protein gene from the winter flounder. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene (wflAFP-6) was integrated into the salmon genome. From a transgenic founder (# 1469), an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish. The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor protein in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. In addition, the antifreeze protein precursor protein level was found to vary with the season, being highest in the month of November and lowest in May. This study had demonstrated a tissue-specific and stable expression of the antifreeze protein transgene in the F3 generation of the transgenic salmon 1469 line.     

Ice-binding mechanism of winter flounder antifreeze proteins

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

Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein.

The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys-Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an alphahelix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain chi1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence-specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.     

Structure-function relationships in a winter flounder antifreeze polypeptide. I. Stabilization of an alpha-helical antifreeze polypeptide by charged-group and hydrophobic interactions

The major antifreeze polypeptide (AFP) from winter flounder (37 amino acid residues) is a single alpha-helix. Aspartic acid and arginine are found, respectively, at the amino and carboxyl-termini. These charged amino acids are ideally located for stabilizing the alpha-helical conformation of this AFP by means of charge-dipole interaction (Shoemaker, K. R., Kim, P.S., York, E.J., Stewart, J. M., and Baldwin, R. L. (1987) Nature 326, 563-567). In order to understand these and other molecular interactions that maintain the AFP structure, we have carried out the chemical synthesis of AFP analogs and evaluated their conformations by circular dichroism spectroscopy. We synthesized the entire AFP molecule (37-mer) and six COOH-terminal peptide fragments (36-, 33-, 27-, 26-, 16-, and 15-mers). Peptides containing acidic NH2-terminal residues displayed greater helix formation and thermal stability compared to those peptides of similar size, but with neutral NH2-terminal residues. Helix formation was maximum above pH 9.2. The peptide conformations also displayed a pH-dependent sensitivity to changes in ionic strength. Helix formation was reduced in the presence of acetonitrile. We conclude that the AFP helix is most likely stabilized by: charge-dipole interactions between charged terminal amino acids and the helix dipole, a charge interaction between Lys18 and Glu22 (either a salt bridge or a hydrogen bond), and hydrophobic interactions.     

The effect of enhanced alpha-helicity on the activity of a winter flounder antifreeze polypeptide.

The antifreeze polypeptide (AFP) from the winter flounder displays partial alpha-helix formation at lower temperatures. To investigate the relationship between antifreeze activity and alpha-helical structure, we designed and then chemically synthesized an AFP analog with enhanced alpha-helicity, and compared its conformation and antifreeze properties with those of the native AFP. The synthetic analog was more helical than the native AFP; however, the antifreeze activity of both peptides were identical. The antifreeze activity of the peptides displayed a strong pH dependence, which paralleled pH-induced changes in helix content. At pH 8.5, the antifreeze activity of both peptides displayed identical concentration dependences. In addition to antifreeze activity measurements, the effects of the peptides on the rate of ice crystal growth were also measured. While both peptides affected the a- and c-axis growth rates of ice crystals, the highly helical analog was able to exert its effect on ice crystal growth rates at 7-8-fold lower concentrations than the native AFP. These data indicate that there is a direct but complex relationship between alpha-helicity and antifreeze activity.     

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.     

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.     

Evidence that translational control mechanisms operate to optimize antifreeze protein production in the winter flounder

In fall and winter, the liver of the winter flounder produces large amounts of alanine-rich (60 mol %) antifreeze proteins for export to the circulation. We have examined the tRNA in the liver to see if the seasonal production of antifreeze protein is accompanied by changes in tRNAAla isoacceptors. Total tRNA from the liver of winter fish showed an approximate 40% increase in alanine acceptor capacity over tRNA from summer fish. In contrast, the acceptor capacities for other amino acids showed no seasonal difference. When labeled alanyl-tRNAs were separated by reverse phase chromatography-5 chromatography, a large proportion of the increase in alanine acceptor capacity was in one of three main peaks. Measurements of the optimum temperatures for various flounder amino-acyl-tRNA synthetases suggest that alanyl-tRNA synthetase functions best between 0 and 5 degrees C, which is the sea water temperature when antifreeze protein synthesis occurs, while prolyl- and valyl-tRNA synthetases are most active between 20 and 30 degrees C. These differences in temperature optima and the seasonal variation in tRNAAla levels and isoaccepting species may both serve to optimize antifreeze protein production by increasing the translational efficiency of its mRNA.     

Hyperactive antifreeze protein from winter flounder is a very long rod-like dimer of alpha-helices

The winter flounder (Pseudopleuronectes americanus) produces short, monomeric alpha-helical antifreeze proteins (type I AFP), which adsorb to and inhibit the growth of ice crystals. These proteins alone are not sufficiently active to protect this fish against freezing at -1.9 degrees C, the freezing point of seawater. We have recently isolated a hyperactive antifreeze protein from the plasma of the flounder with activity 10-100-fold higher than type I AFP. It is comparable in activity to the AFPs produced by insects, and is capable of conferring freeze resistance to the flounder. This novel AFP has a molecular mass of 16,683 Da and a remarkable amino acid composition that is >60% alanine. CD spectra indicate that the protein is almost entirely alpha-helical at 4 degrees C but partially denatures at 20 degrees C, resulting in a species with a moderately reduced helix content that is stable at up to 50 degrees C. This transformation correlates with irreversible loss of activity. Analytical ultracentrifugation (sedimentation velocity and equilibrium) indicates that the predominant species in solution is dimeric (molecular weight, 32,275). Size-exclusion chromatography reveals a 2-fold higher apparent molecular weight suggesting that this molecule has an unusually large Stokes radius. The axial ratio of the dimer calculated from the sedimentation velocity data is 18:1, confirming that this protein has an extraordinarily long, rod-like structure, consistent with a novel dimeric alpha-helical arrangement. The structural model that best fits these data is one in which the approximately 195 amino acids of each monomer form one approximately 290-A long alpha-helix and associate via a unique dimerization motif that is distinct from that of the leucine zipper and any other coiled-coil.     

Intermolecular interaction studies of winter flounder antifreeze protein reveal the existence of thermally accessible binding state

The physical nature underlying intermolecular interactions between two rod-like winter flounder antifreeze protein (AFP) molecules and their implication for the mechanism of antifreeze function are examined in this work using molecular dynamics simulations, augmented with free energy calculations employing a continuum solvation model. The energetics for different modes of interactions of two AFP molecules is examined in both vacuum and aqueous phases along with the water distribution in the region encapsulated by two antiparallel AFP backbones. The results show that in a vacuum two AFP molecules intrinsically attract each other in the antiparallel fashion, where their complementary charge side chains face each other directly. In the aqueous environment, this attraction is counteracted by both screening and entropic effects. Therefore, two nearly energetically degenerate states, an aggregated state and a dissociated state, result as a new aspect of intermolecular interaction in the paradigm for the mechanism of action of AFP. The relevance of these findings to the mechanism of function of freezing inhibition in the context of our work on Antarctic cod antifreeze glycoprotein (Nguyen et al., Biophysical Journal, 2002, Vol. 82, pp. 2892-2905) is discussed.     

Biosynthesis, import and processing of precursor polypeptides of mammalian mitochondrial pyruvate dehydrogenase complex.

An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be observed in uncoupler-treated BRL or NBL-1 cells. Pre-E1 alpha, pre-E1 beta and also pre-E3, have signal sequences in the Mr range 1500-3000 while pre-E2 contains a long additional segment of Mr 7000-9000. All of these forms exhibit similar kinetics of processing to the mature subunits with a transit time of 10-12 min. In NBL-1 cells, E3 is present in the immune complexes formed with anti-PDC serum whereas this is not the case in PK-15 cells. Thus, there are significant variations in the affinity of lipoamide dehydrogenase (E3) for the E2 core structure in different species. Pre-E1 alpha accumulates only poorly in PK-15 cells and is aberrantly processed on removal of uncoupler. This precursor is markedly more stable in NBL-1 and BRL cells. The lack of detection of a precursor form of component X is also discussed.     

The effect of hydrophobic analogues of the type I winter flounder antifreeze protein on lipid bilayers

The effect of four synthetic analogues of the 37-residue winter flounder type I antifreeze protein (AFP), which contain four Val, Ala or Ile residues in place of Thr residues at positions 2, 13, 24 and 37 and two additional salt bridges, on the binary lipid system prepared from a 1:1 mixture of the highly unsaturated DGDG and saturated DMPC has been determined using FTIR spectroscopy. In contrast to the natural protein, which increases the thermotropic phase transition, the Thr, Val and Ala analogues decreased the thermotropic phase transitions of the liposomes by 2.2 degrees Celsius, 3.4 degrees Celsius and 2.4 degrees Celsius, while the Ile analogue had no effect on the transition. Experiments performed using perdeuterated DMPC showed that the Ala and Thr peptides interacted preferentially with the DGDG in the lipid mixture, while the Val peptide showed no preference for either lipid. The results are consistent with interactions involving the hydrophobic face of type I AFPs and model bilayers, i.e. the same face of the protein that is responsible for antifreeze properties. The different effects correlate with the helicity of the peptides and suggest that the solution conformation of the peptides has a significant role in determining the effects of the peptides on thermotropic membrane phase transitions.     

Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli

HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function.