A two-dimensional adsorption kinetic model for thermal hysteresis activity in antifreeze proteins.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), collectively abbreviated as AF(G)Ps, are synthesized by various organisms to enable their cells to survive in subzero environments. Although the AF(G)Ps are markedly diverse in structure, they all function by adsorbing to the surface of embryonic ice crystals to inhibit their growth. This adsorption results in a freezing temperature depression without an appreciable change in the melting temperature. The difference between the melting and freezing temperatures, termed thermal hysteresis (TH), is used to detect and quantify the antifreeze activity. Insights from crystallographic structures of a number of AFPs have led to a good understanding of the ice-protein interaction features. Computational studies have focused either on verifying a specific model of AFP-ice interaction or on understanding the protein-induced changes in the ice crystal morphology. In order to explain the origin of TH, we propose a novel two-dimensional adsorption kinetic model between AFPs and ice crystal surfaces. The validity of the model has been demonstrated by reproducing the TH curve on two different beta-helical AFPs upon increasing the protein concentration. In particular, this model is able to accommodate the change in the TH behavior observed experimentally when the size of the AFPs is increased systematically. Our results suggest that in addition to the specificity of the AFPs for the ice, the coverage of the AFPs on the ice surface is an equally necessary condition for their TH activity.

Antifreeze proteins differentially affect model membranes during freezing.

Over the past decade antifreeze proteins from polar fish have been shown either to stabilize or disrupt membrane structure during low temperature and freezing stress. However, there has been no systematic study on how membrane composition affects the interaction of antifreeze proteins with membranes under stress conditions. Therefore, it is not possible at present to predict which antifreeze proteins will protect, and which will damage a particular membrane during chilling or freezing. Here, we analyze the effects of freezing on spinach thylakoid membranes and on model membranes of varying lipid composition in the presence of antifreeze protein type I (AFP I) and specific fractions of antifreeze glycoproteins (AFGP). We find that the addition of galactolipids to phospholipid model membranes changes the effect each protein has on the membrane during freezing. However, the greatest differences observed in this study are between the different types of antifreeze proteins. We find that AFP type I and the largest molecular weight fractions of AFGP induce concentration dependent leakage from, and are fusogenic to the liposomes. This is the first report that an antifreeze protein induces membrane fusion. In contrast, the smallest fraction of AFGP offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to 10 mg/ml.

Comparison of the solution conformation and dynamics of antifreeze glycoproteins from Antarctic fish.

The (1)H- and (13)C-NMR spectra of antifreeze glycoprotein fractions 1-5 from Antarctic cod have been assigned, and the dynamics have been measured using (13)C relaxation at two temperatures. The chemical shifts and absence of non-sequential (1)H-(1)H NOEs are inconsistent with a folded, compact structure. (13)C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1-5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555-1563). The infrared (IR) spectra of AFGP8 and AFGP1-5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1-5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible o, psi angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops.

Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.

Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.

Antifreeze glycoproteins (AFGPs), found in the blood of polar fish at concentrations as high as 35 g/liter, are known to prevent ice crystal growth and depress the freezing temperature of the blood. Previously, Rubinsky et al. [Rubinsky, B., Mattioli, M., Arav, A., Barboni, B. & Fletcher, G. L. (1992) Am. J. Physiol. 262, R542-R545] provided evidence that AFGPs block ion fluxes across membranes during cooling, an effect that they ascribed to interactions with ion channels. We investigated the effects of AFGPs on the leakage of a trapped marker from liposomes during chilling. As these liposomes are cooled through the transition temperature, they leak approximately 50% of their contents. Addition of less than 1 mg/ml of AFGP prevents up to 100% of this leakage, both during chilling and warming through the phase transition. This is a general effect that we show here applies to liposomes composed of phospholipids with transition temperatures ranging from 12 degrees C to 41 degrees C. Because these results were obtained with liposomes composed of phospholipids alone, we conclude that the stabilizing effects of AFGPs on intact cells during chilling reported by Rubinsky et al. may be due to a nonspecific effect on the lipid components of native membranes. There are other proteins that prevent leakage, but only under specialized conditions. For instance, antifreeze proteins, bovine serum albumin, and ovomucoid all either have no effect or actually induce leakage. Following precipitation with acetone, all three proteins inhibited leakage, although not to the extent seen with AFGPs. Alternatively, there are proteins such as ovotransferrin that have no effect on leakage, either before or after acetone precipitation.

The development and evolution of U.S. Army field rations.

Armed conflicts create large masses of personnel that need to be fed nutritious foods in less than favorable conditions. The United States military, private industry, and academia continue to work together to develop rations that meet the needs of military personnel who find themselves in varying climates, conditions, and geography.

Effect of boronic acids on antifreeze proteins.

The activity of antifreeze glycoprotein from the blood serum of Boreagadus saida was strongly inhibited by ions of organic boronic acids as well as by borate. The activity of nonglycoprotein from the blood serum of Pseudopleuronectus americanus, however, was not similarly inhibited. The inhibition by borate is thus specific for molecules with the carbohydrate moiety.

Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases.

This paper presents kinetic properties of the transfer of several synthetic 9-substituted sialic acid analogues onto N- or O-linked glycoprotein glycans by four purified mammalian sialyltransferases: Gal beta 1,4GlcNac alpha 2,6sialyltransferase, Gal beta-1,4(3)GlcNAc alpha 2,3-sialyltransferase, Gal beta 1,3GalNAc alpha 2,3sialyltransferase, and GalNAc alpha 2,6sialyltransferase. The substituents at C-9 of the sialic acid analogues introduce special biochemical characteristics: 9-Amino-NeuAc represents, up to the present, the first derivative that is resistant toward bacterial, viral, and mammalian sialidases but is transferred by a sialyltransferase. 9-Acetamido-NeuAc, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc differ in size and hydrophobic character from each other and from parent NeuAc. 9-Azido-NeuAc may be used to introduce a photoreactive label. The kinetic properties of the four sialyltransferases with regard to the donor CMP-glycosides differed distinctly depending on the structure of the substituent at C-9. CMP-9-amino-NeuAc was only accepted as donor substrate by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase (rat liver), but the Km value was 14-fold higher than that of parent CMP-NeuAc. In contrast, 9-azido-NeuAc was readily transferred by each of these four enzymes. 9-Acetamido-NeuAc, which is a receptor analogue for influenza C virus, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc were also accepted by each sialyltransferase, but incorporation values differed significantly depending on the enzyme used. For the first time, the resialylation of asialo-alpha 1-acid glycoprotein with 9-substituted sialic acid analogues by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase is demonstrated.

Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins.

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (alpha-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was greater than 70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)2, 88; (Gly)3, 82; (Gly)4, 70; Gly-Gly-NH2, 44; Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.

Egg-white and blood-serum proteins functioning by noncovalent interactions: studies by chemical modification and comparative biochemistry.

Some of the more interesting and important proteins are those that function by forming associations or complexes with other substances. The structure-function relationships of three of these with very different substances are transferrins, which chelate metal ions; avian ovomucoids, which form complexes with proteolytic enzymes; and antifreeze glycoproteins, which interact at the ice-solution interface. Interrelating studies on the comparative biochemistry with studies using chemical modification have helped identify the side-chain groups of the proteins involved in function as well as to be useful for studies on general protein chemistry. The most strongly associated interaction is the chelation of iron by transferrin, with an association constant of approximately 10(21); tyrosines, histidines, and sometimes aspartate are involved. For ovomucoids, individual substratelike residues such as lysine are involved in a Michaelis-like complex, and association constants are as high as 10(10). By contrast, the antifreeze glycoproteins appear to function by a polymeric interaction at the surface of ice, with a much weaker association.

Reflections on chemical modification of proteinases and their protein inhibitors.

Proteolytic enzymes and their protein inhibitors have been studied by chemical modification for over four decades. Modifications have helped to identify the active (and reactive) sites and to understand their mechanisms of interaction. Inactive derivatives of the enzymes form stable complexes with some inhibitors. These inactive enzymes have also been used for affinity chromatographic adsorptions.

Ice growth in supercooled solutions of antifreeze glycoprotein.

Inhibition of ice growth in supercooled solution by certain proteins is vital to the survival of many living organisms. Some fish, native to both subzero northern and southern waters, have special proteins or glycoproteins in their blood serum that inhibit ice formation. Whereas these proteins have only a very small effect on the melting temperature of ice, the temperature of these fish can fall to nearly 1 K below the melting point before ice crystals grow. This phenomenon is called freezing hysteresis, in contrast to the normal colligative effect of solutes that depresses the equilibrium temperature, around which small changes lead to crystal growth or melting depending on sign. Some insects also exhibit a serum freezing hysteresis. We report the effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of these antifreeze glycoproteins (AFGPs). We find that the crystallization rate is up to five times greater than that in pure water.

Chemical modification of proteins: comments and perspectives.

The use of chemical modification of proteins has increased exponentially during the past two decades. Today the many different uses of chemical modification include determination of relative reactivities of side chain groups, the quantitation of individual amino acids, development of affinity reagents, mechanism-based reagents for pharmaceutical uses, cross-linking reagents, special techniques for bioprostheses, blocking reagents for peptide synthesis, and reagents for specific cleavages of peptide bonds. Chemical modification should continue to be a primary tool in protein chemistry. It can supply information or products difficult or impossible to attain by the newer powerful technique of in vitro mutagenesis as well as serve as a supplementary procedure for the latter.

The interaction of anions with native and phenylglyoxal-modified human serum transferrin.

The interaction of various anions with human serum transferrin was investigated due to the concomitant binding of iron and a synergistic anion to form the transferrin-anion-iron complex. Two tetrahedral oxyanion oxidizing agents, periodate and permanganate, were found to partially inactivate transferrin when used at equimolar ratios of oxidizing agent to protein active sites. Hypochlorite, a strong oxidizing agent with little structural similarity to periodate and permanganate, had little effect on iron-binding activity when used at similar low molar ratios of reagent to transferrin active sites. Transferrin treated with a 3:1 molar ratio of periodate or permanganate to active sites lost 74 or 67% of its iron-binding capacity, respectively. The composition of the buffer affected the extent of transferrin inactivation by periodate and permanganate; for example, the extent of inactivation by periodate was threefold greater in a borate buffer than in a phosphate buffer. Comparative oxidations in buffer systems suggest the following order of affinity of three buffer anions for the apotransferrin metal-binding center: phosphate greater than bicarbonate greater than borate. The interaction of phosphate ions with the iron-transferrin complex was also examined due to the increased susceptibility to periodate inactivation of iron-saturated transferrin in phosphate buffer (M. H. Penner, R. B. Yamasaki, D. T. Osuga, D. R. Babin, C. F. Meares, and R. E. Feeney (1983) Arch. Biochem. Biophys. 225, 740-747). The apparent destabilization of the iron-transferrin complex in phosphate buffer was found to be due to the competitive removal of iron by phosphate from the iron-protein complex. We found that phenylglyoxal-modified Fe-transferrin, with no loss of bound iron, was much more resistant to iron removal by phosphate and other competitive chelators.

Conformation of the glycotripeptide repeating unit of antifreeze glycoprotein of polar fish as determined from the fully assigned proton n.m.r. spectrum.

With its simple glycotripeptide repeating structure the antifreeze glycoprotein of polar fish may be an especially simple conformational mode for mucin glycoproteins with similar but more complex structures. The fully assigned proton n.m.r. spectrum confirms the anomeric configurations of the hexapyranosidic sugars of the side chains and the coupling constants of the alpha GalNAc and the beta Gal residues show both to be in the expected 4C1 chair conformation. The assignment of a single resonance for each proton of the (Ala-Thr-Ala)n repeat unit coupled with the observation of long range nuclear Overhauser effects (n.O.e.) implies a three-fold repeating conformation. The resonances of the two alanines are distinct and can be assigned to their correct positions in the peptide sequence by n.O.e. observed at the amide proton resonances on saturation of the alpha proton signals. The amide proton coupling constants of all three peptide residues are similar and imply a limited range of peptide backbone torsion angles, phi CN. The large n.O.e. which has been observed between the amide proton and the alpha proton of the residue preceding it in the sequence implies large positive values for the peptide dihedral angle, psi CC. Limits are placed on possible values of side chain dihedral angles by the observation of the coupling constant between the alpha and beta protons of the threonyl residue. The observation of n.O.e. between the anomeric proton of GalNAc and the threonyl side chain protons gives information on the conformation of the alpha glycosidic linkage between the disaccharide and the peptide. n.O.e. observed between the protons of the beta glycosidic linkage indicates the conformation of the disaccharide and the large amide proton coupling constant of the GalNAc residue shows a trans proton relationship. The spectroscopically derived data have been combined with conformational energy calculations to give a conformational model for antifreeze glycoprotein in which the hydrophobic surfaces of the disaccharide side chains are wrapped closely against a three-fold left handed helical peptide backbone. The hydrophilic sides of the disaccharides are aligned so that they may bind to the ice crystal face, which is perpendicular to the fast growth axis inhibiting normal crystal growth.

Purification and primary sequences of the major arginine-containing antifreeze glycopeptides from the fish Eleginus gracilis.

An Arg-containing antifreeze glycoprotein from the polar fish Eleginus gracilis was isolated, and the major components were purified to homogeneity. The general protocol for purification was chromatography of serum on DEAE-cellulose, followed by chromatography on a cation exchanger. DEAE-cellulose chromatography resulted in two fractions, A and B. Fraction A contained most of the antifreeze glycoprotein found in E. gracilis (approximately 80% by weight) and consisted of 13 distinct components. Unlike antifreeze glycoproteins from other previously studied polar fish, Fraction A contained both low and high molecular weight antifreeze glycoprotein components. The two major components of Fraction A were sequenced and compared with the sequence of antifreeze glycoproteins 7 and 8 from both Boreogadus saida and Pagothenia borchgrevinki. The antifreeze glycoproteins from E. gracilis were shown to have a similar composition to those previously studied, except for an additional Ala-Arg dipeptide at the carbon terminal in the major components of Fraction A and the position of Pro in the low molecular weight components. The activity of E. gracilis antifreeze glycoproteins is the subject of a companion article (Burcham, T. S., Osuga, D. T., Yeh, Y., and Feeney, R. E. (1986) J. Biol. Chem. 261, 6390-6397).

A kinetic description of antifreeze glycoprotein activity.

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.