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Brian D. Sykes - One of the best experts on this subject based on the ideXlab platform.
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enhancing the activity of a β helical Antifreeze Protein by the engineered addition of coils
Biochemistry, 2004Co-Authors: Christopher B. Marshall, Margaret E Daley, Brian D. Sykes, Peter L DaviesAbstract:The effectiveness of natural Antifreeze Proteins in inhibiting the growth of a seed ice crystal seems to vary with Protein size. Here we have made use of the extreme regularity of the β-helical Antifreeze Protein from the beetle Tenebrio molitor to explore systematically the relationship between Antifreeze activity and the area of the ice-binding site. Each of the 12-amino acid, disulfide-bonded central coils of the β-helix contains a Thr-Xaa-Thr ice-binding motif. By adding coils to, and deleting coils from, the seven-coil parent Antifreeze Protein, we have made a series of constructs with 6−11 coils. Misfolded forms of these Antifreezes were removed by ice affinity purification to accurately compare the specific activity of each construct. There was a 10−100-fold gain in activity upon going from six to nine coils, depending on the concentration that was compared. Activity was maximal for the nine-coil construct, which gave a freezing point depression of 6.5 C° at 0.7 mg/mL, but actually decreased for th...
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Characterization of threonine side chain dynamics in an Antifreeze Protein using natural abundance ^13C NMR spectroscopy
Journal of Biomolecular NMR, 2004Co-Authors: Margaret E Daley, Brian D. SykesAbstract:The dynamics of threonine side chains of the Tenebrio molitor Antifreeze Protein (TmAFP) were investigated using natural abundance ^13C NMR. In TmAFP, the array of threonine residues on one face of the Protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the ^1H-^13C NOE were determined in this study. The CαH relaxation measurements were compared to the previously measured ^15N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the χ_1 dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc. , 100 , 7159–7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than ±25°.
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the role of side chain conformational flexibility in surface recognition by tenebrio molitor Antifreeze Protein
Protein Science, 2003Co-Authors: Margaret E Daley, Brian D. SykesAbstract:Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β-helical Tenebrio molitor Antifreeze Protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H-1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the Protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand.
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identification of the ice binding face of Antifreeze Protein from tenebrio molitor
FEBS Letters, 2002Co-Authors: Christopher B. Marshall, Margaret E Daley, Brian D. Sykes, Laurie A Graham, Peter L DaviesAbstract:The beetle Tenebrio molitor produces several isoforms of a highly disulfide-bonded β-helical Antifreeze Protein with one surface comprised of an array of Thr residues that putatively interacts with ice. In order to use mutagenesis to identify the ice-binding face, we have selected an isoform that folds well and is tolerant of amino acid substitution, and have developed a heating test to monitor refolding. Three different types of steric mutations made to the putative ice-binding face reduced thermal hysteresis activity substantially while a steric mutation on an orthogonal surface had little effect. NMR spectra indicated that all mutations affected Protein folding to a similar degree and demonstrated that most of the Protein folded well. The large reductions in activity associated with steric mutations in the Thr array strongly suggest that this face of the Protein is responsible for ice binding.
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β helix structure and ice binding properties of a hyperactive Antifreeze Protein from an insect
Nature, 2000Co-Authors: Brian D. Sykes, Virginia K. Walker, Michael J. Kuiper, Steffen P Graether, Stephane M Gagne, Zongchao Jia, Peter L DaviesAbstract:β-Helix structure and ice-binding properties of a hyperactive Antifreeze Protein from an insect
Peter L Davies - One of the best experts on this subject based on the ideXlab platform.
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an Antifreeze Protein folds with an interior network of more than 400 semi clathrate waters
Science, 2014Co-Authors: Tianjun Sun, Fenghsu Lin, Robert L Campbell, John S Allingham, Peter L DaviesAbstract:When polypeptide chains fold into a Protein, hydrophobic groups are compacted in the center with exclusion of water. We report the crystal structure of an alanine-rich Antifreeze Protein that retains ~400 waters in its core. The putative ice-binding residues of this dimeric, four-helix bundle Protein point inwards and coordinate the interior waters into two intersecting polypentagonal networks. The bundle makes minimal Protein contacts between helices, but is stabilized by anchoring to the semi-clathrate water monolayers through backbone carbonyl groups in the Protein interior. The ordered waters extend outwards to the Protein surface and likely are involved in ice binding. This Protein fold supports both the anchored-clathrate water mechanism of Antifreeze Protein adsorption to ice and the water-expulsion mechanism of Protein folding.
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Solution structure of hyperactive type I Antifreeze Protein
RSC Adv., 2013Co-Authors: Luuk L. C. Olijve, Peter L Davies, Tianjun Sun, Theyencheri Narayanan, Corinne Jud, Ilja K. VoetsAbstract:Antifreeze Proteins (AFPs) protect freeze-intolerant fish species living in icy polar waters against freeze damage. A 34 kDa dimeric type I Antifreeze Protein (wfAFP 1h) with unusually high activity in comparison to all other Antifreeze Proteins in fish was recently discovered in the winter flounder. We have measured the size and shape of this hyperactive AFP by using small angle X-ray scattering. Our results show that wfAFP 1h adopts a long cylindrical shape with a length of 19 ± 2 nm and a diameter of 2.3 ± 0.2 nm, which means that wfAFP 1h does not form a fully extended helical dimer in solution. These findings call for a revision of the structural model of wfAFP 1h and the concept of a flat, threonine-rich ice-binding site extending down the length of the Protein. Instead, the hyperactive nature of wfAFP 1h may be derived from a unique 3D arrangement of the helices —yet to be resolved— by which it is able to bind to ice surfaces.
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compound ice binding site of an Antifreeze Protein revealed by mutagenesis and fluorescent tagging
Biochemistry, 2010Co-Authors: Christopher P Garnham, Ido Braslavsky, Adam J Middleton, Aditya Natarajan, Mike J Kuiper, Peter L DaviesAbstract:By binding to the surface of ice crystals, type III Antifreeze Protein (AFP) can depress the freezing point of fish blood to below that of freezing seawater. This 7-kDa globular Protein is encoded by a multigene family that produces two major isoforms, SP and QAE, which are 55% identical. Disruptive mutations on the ice-binding site of type III AFP lower Antifreeze activity but can also change ice crystal morphology. By attaching green fluorescent Protein to different mutants and isoforms and by examining the binding of these fusion Proteins to single-crystal ice hemispheres, we show that type III AFP has a compound ice-binding site. There are two adjacent, flat, ice-binding surfaces at 150° to each other. One binds the primary prism plane of ice; the other, a pyramidal plane. Steric mutations on the latter surface cause elongation of the ice crystal as primary prism plane binding becomes dominant. SP isoforms naturally have a greatly reduced ability to bind the prism planes of ice. Mutations that make th...
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identification of the ice binding face of a plant Antifreeze Protein
FEBS Letters, 2009Co-Authors: Adam J Middleton, Peter L Davies, Alan Brown, Virginia K. WalkerAbstract:The Antifreeze Protein of Lolium perenne, a perennial ryegrass, was previously modeled as a beta-roll with two extensive flat beta-sheets on opposite sides of the molecule. Here we have validated the model with a series of nine site-directed steric mutations in which outward-pointing short side-chain residues were replaced by tyrosine. None of these disrupted the fold. Mutations on one of the beta-sheets and on the sides of the Protein retained 70% or greater activity. Three mutations that clustered on the other flat surface lost up to 90% of their Antifreeze activity and identify this beta-sheet as the ice-binding face.
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growth melt asymmetry in ice crystals under the influence of spruce budworm Antifreeze Protein
Journal of Physics: Condensed Matter, 2007Co-Authors: Natalya Pertaya, Peter L Davies, Yeliz Celik, Carlos L Diprinzio, J S Wettlaufer, Ido BraslavskyAbstract:Here we describe studies of the crystallization behavior of ice in an aqueous solution of spruce budworm Antifreeze Protein (sbwAFP) at atmospheric pressure. SbwAFP is an ice binding Protein with high thermal hysteresis activity, which helps protect Choristoneura fumiferana (spruce budworm) larvae from freezing as they overwinter in the spruce and fir forests of the north eastern United States and Canada. Different types of ice binding Proteins have been found in many other species. They have a wide range of applications in cryomedicine and cryopreservation, as well as the potential to protect plants and vegetables from frost damage through genetic engineering. However, there is much to learn regarding the mechanism of action of ice binding Proteins. In our experiments, a solution containing sbwAFP was rapidly frozen and then melted back, thereby allowing us to produce small single crystals. These maintained their hexagonal shapes during cooling within the thermal hysteresis gap. Melt– growth–melt sequences in low concentrations of sbwAFP reveal the same shape transitions as are found in pure ice crystals at low temperature (−22 ◦ C) and high pressure (2000 bar) (Cahoon et al 2006 Phys. Rev. Lett. 96 255502); while both growth and melt shapes display faceted hexagonal morphology, they are rotated 30 ◦ relative to one another. Moreover, the initial melt shape and orientation is recovered in the sequence. To visualize the binding of sbwAFP to ice, we labeled the Antifreeze Protein with enhanced green fluorescent Protein (eGFP) and observed the sbwAFP–GFP molecules directly on ice crystals using confocal microscopy. When cooling the ice crystals, facets form on the six primary prism planes (slowest growing planes) that are evenly decorated with sbwAFP–GFP. During melting, apparent facets form on secondary prism planes (fastest melting planes), leaving residual sbwAFP at the six corners of the
Margaret E Daley - One of the best experts on this subject based on the ideXlab platform.
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enhancing the activity of a β helical Antifreeze Protein by the engineered addition of coils
Biochemistry, 2004Co-Authors: Christopher B. Marshall, Margaret E Daley, Brian D. Sykes, Peter L DaviesAbstract:The effectiveness of natural Antifreeze Proteins in inhibiting the growth of a seed ice crystal seems to vary with Protein size. Here we have made use of the extreme regularity of the β-helical Antifreeze Protein from the beetle Tenebrio molitor to explore systematically the relationship between Antifreeze activity and the area of the ice-binding site. Each of the 12-amino acid, disulfide-bonded central coils of the β-helix contains a Thr-Xaa-Thr ice-binding motif. By adding coils to, and deleting coils from, the seven-coil parent Antifreeze Protein, we have made a series of constructs with 6−11 coils. Misfolded forms of these Antifreezes were removed by ice affinity purification to accurately compare the specific activity of each construct. There was a 10−100-fold gain in activity upon going from six to nine coils, depending on the concentration that was compared. Activity was maximal for the nine-coil construct, which gave a freezing point depression of 6.5 C° at 0.7 mg/mL, but actually decreased for th...
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Characterization of threonine side chain dynamics in an Antifreeze Protein using natural abundance ^13C NMR spectroscopy
Journal of Biomolecular NMR, 2004Co-Authors: Margaret E Daley, Brian D. SykesAbstract:The dynamics of threonine side chains of the Tenebrio molitor Antifreeze Protein (TmAFP) were investigated using natural abundance ^13C NMR. In TmAFP, the array of threonine residues on one face of the Protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the ^1H-^13C NOE were determined in this study. The CαH relaxation measurements were compared to the previously measured ^15N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the χ_1 dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc. , 100 , 7159–7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than ±25°.
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the role of side chain conformational flexibility in surface recognition by tenebrio molitor Antifreeze Protein
Protein Science, 2003Co-Authors: Margaret E Daley, Brian D. SykesAbstract:Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β-helical Tenebrio molitor Antifreeze Protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H-1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the Protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand.
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identification of the ice binding face of Antifreeze Protein from tenebrio molitor
FEBS Letters, 2002Co-Authors: Christopher B. Marshall, Margaret E Daley, Brian D. Sykes, Laurie A Graham, Peter L DaviesAbstract:The beetle Tenebrio molitor produces several isoforms of a highly disulfide-bonded β-helical Antifreeze Protein with one surface comprised of an array of Thr residues that putatively interacts with ice. In order to use mutagenesis to identify the ice-binding face, we have selected an isoform that folds well and is tolerant of amino acid substitution, and have developed a heating test to monitor refolding. Three different types of steric mutations made to the putative ice-binding face reduced thermal hysteresis activity substantially while a steric mutation on an orthogonal surface had little effect. NMR spectra indicated that all mutations affected Protein folding to a similar degree and demonstrated that most of the Protein folded well. The large reductions in activity associated with steric mutations in the Thr array strongly suggest that this face of the Protein is responsible for ice binding.
Virginia K. Walker - One of the best experts on this subject based on the ideXlab platform.
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identification of the ice binding face of a plant Antifreeze Protein
FEBS Letters, 2009Co-Authors: Adam J Middleton, Peter L Davies, Alan Brown, Virginia K. WalkerAbstract:The Antifreeze Protein of Lolium perenne, a perennial ryegrass, was previously modeled as a beta-roll with two extensive flat beta-sheets on opposite sides of the molecule. Here we have validated the model with a series of nine site-directed steric mutations in which outward-pointing short side-chain residues were replaced by tyrosine. None of these disrupted the fold. Mutations on one of the beta-sheets and on the sides of the Protein retained 70% or greater activity. Three mutations that clustered on the other flat surface lost up to 90% of their Antifreeze activity and identify this beta-sheet as the ice-binding face.
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effect of Antifreeze Protein on nucleation growth and memory of gas hydrates
Aiche Journal, 2006Co-Authors: Huang Zeng, Igor L Moudrakovski, John A Ripmeester, Virginia K. WalkerAbstract:The effect of Type I Antifreeze Protein (AFP) from winter flounder on the formation of propane hydrate and methane hydrate was studied. We show that the formation of both hydrates is inhibited significantly, with both nucleation and crystal growth being affected. Also, AFP showed the so-far unique ability to eliminate the "memory effect" in the reformation of gas hydrate. We have proposed a mechanism involving the interference of AFP with heterogeneous nucleation and subsequent growth of the hydrates. It is also shown that a number of samples must be studied in order to obtain meaningful statistics, and that magnetic resonance imaging provides a novel way of studying the nucleation and growth of hydrate in multiple droplets.
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the inhibition of tetrahydrofuran clathrate hydrate formation with Antifreeze Protein
Canadian Journal of Physics, 2003Co-Authors: Huang Zeng, Virginia K. Walker, Lee D Wilson, John A RipmeesterAbstract:The effect of Type I fish Antifreeze Protein (AFP) from the winter flounder, Pleuronectes americanus (Walbaum), (WfAFP) on the formation of tetrahydrofuran (THF) clathrate hydrate was studied by observing changes in THF crystal morphology and determining the induction time for nucleation. AFP retarded THF clathrate-hydrate growth at the tested temperatures and modified the THF clathrate-hydrate crystal morphology from octahedral to plate-like. AFP appears to be even more effective than the kinetic inhibitor, polyvinylpyrrolidone (PVP). Recombinant AFP from an insect, a spruce budworm, Choristoneura fumiferana (Clem.), moth, (Cf) was also tested for inhibition activity by observation of the THF-hydrate-crystal-growth habit. Like WfAFP, CfAFP appeared to show adsorption on multiple THF-hydrate-crystal faces. A Protein with no Antifreeze activity, cytochrome C, was used as a control and it neither changed the morphology of the THF clathrate-hydrate crystals, nor retarded the formation of the hydrate. Prelimi...
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a β helical Antifreeze Protein isoform with increased activity structural and functional insights
Journal of Biological Chemistry, 2002Co-Authors: Eeva K Leinala, Peter L Davies, Virginia K. Walker, Daniel Doucet, Michael G Tyshenko, Zongchao JiaAbstract:Abstract The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active Antifreeze Protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular β-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the Protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Δ-2-loop, results in a Protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced Antifreeze activity of CfAFP-501 is directly correlated to the length of its β-helical structure and hence the size of its ice-binding face.
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β helix structure and ice binding properties of a hyperactive Antifreeze Protein from an insect
Nature, 2000Co-Authors: Brian D. Sykes, Virginia K. Walker, Michael J. Kuiper, Steffen P Graether, Stephane M Gagne, Zongchao Jia, Peter L DaviesAbstract:β-Helix structure and ice-binding properties of a hyperactive Antifreeze Protein from an insect
Garth Fletcher - One of the best experts on this subject based on the ideXlab platform.
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Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder Antifreeze Protein gene
Transgenic Research, 1999Co-Authors: Choy L. Hew, Raymond Poon, Fei Xiong, Sherry Gauthier, Margaret Shears, Madonna King, Peter Davies, Garth FletcherAbstract: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.
