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Antifreeze Protein

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Brian D. Sykes – One of the best experts on this subject based on the ideXlab platform.

  • enhancing the activity of a β helical Antifreeze Protein by the engineered addition of coils
    Biochemistry, 2004
    Co-Authors: Christopher B. Marshall, Margaret E Daley, Brian D. Sykes, Peter L Davies

    Abstract:

    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, 2004
    Co-Authors: Margaret E Daley, Brian D. Sykes

    Abstract:

    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, 2003
    Co-Authors: Margaret E Daley, Brian D. Sykes

    Abstract:

    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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Peter L Davies – One of the best experts on this subject based on the ideXlab platform.

  • an Antifreeze Protein folds with an interior network of more than 400 semi clathrate waters
    Science, 2014
    Co-Authors: Tianjun Sun, Fenghsu Lin, Robert L Campbell, John S Allingham, Peter L Davies

    Abstract:

    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., 2013
    Co-Authors: Luuk L. C. Olijve, Peter L Davies, Tianjun Sun, Theyencheri Narayanan, Corinne Jud, Ilja K. Voets

    Abstract:

    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, 2010
    Co-Authors: Christopher P Garnham, Ido Braslavsky, Adam J Middleton, Aditya Natarajan, Mike J Kuiper, Peter L Davies

    Abstract:

    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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Margaret E Daley – One of the best experts on this subject based on the ideXlab platform.

  • enhancing the activity of a β helical Antifreeze Protein by the engineered addition of coils
    Biochemistry, 2004
    Co-Authors: Christopher B. Marshall, Margaret E Daley, Brian D. Sykes, Peter L Davies

    Abstract:

    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, 2004
    Co-Authors: Margaret E Daley, Brian D. Sykes

    Abstract:

    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, 2003
    Co-Authors: Margaret E Daley, Brian D. Sykes

    Abstract:

    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.

    Free Register to Access Article