The redesign of antifreeze proteins: an improved procedure

Jeroen
Vrancken

Everyone has at least once experienced on a hot summer day the melting of their ice cream. To slow down this process, Unilever started introducing antifreeze proteins in their ice cream about ten years ago. Originally, they were derived from ocean pout, but for this industrial process genetically manipulated brewer’s yeast is used to produce higher quantities of these proteins. This animal-friendly method allows Unilever to produce ice cream with a reduced fat content, without a change in the taste, that will retain their solid structure longer. Additionally, with the reduced fat content, it is possible to have a higher fruit content, making the new ice creams even healthier.

Upon further research it was observed that antifreeze proteins, derived from different organisms such as rye-grass and extremophile bacteria, contain a linear structure consisting of repeating units that coordinates on ice crystal lattices. The presence of these repeating units is interesting for the biotechnology for several reasons, but mainly because it gives the possibility to modify these proteins in such a way that identical repeating units are obtained. Once this is done, repeating units can easily be added or deleted, resulting in a set of proteins that only vary in the amount of repeating units. Such a set of proteins has specific purposes in, i.e., the coordination of metal arrays, as the length can be easily modified. For this reason, the conducted lab work focused on these proteins.

During the first part of this research a procedure was developed that uses the sequences of antifreeze proteins as an input to computationally generate the desired proteins. The procedure helps to identify the different repeats, which will be aligned and compared. Via computational methods it was then possible to generate putative ancestral sequences for these repeats, as they probably all share the same evolutionary origin, but diversified due to mutations over the course of time. The different ancestral sequences were assessed on their properties and the most promising repeat sequence was chosen. As such, proteins can be designed with a varying number of identical repeats in its core.

However, no proteins were obtained after multiple attempts to bring the proteins to expression. After investigating the possible reasons as to why no proteins were obtained, alterations were made to the design algorithms and procedure. These changes resulted in an improved procedure that would now combine two repeats and use these to generate multiple alternating repeats instead of multiple identical repeats. This increase in diversity may have a stabilising effect on the protein. After comparing the newly obtained sequences with the initial sequences, it was clear that the new sequences were improved as they clearly had a better design score. However, due to a lack of time these improvements could not be tested experimentally, but only computationally.

In the future, these improved designs will be validated experimentally and, if a set of proteins is obtained, it is possible to assess these proteins based on their antifreeze activity as well. If they still contain their antifreeze activity, they can be compared to the original protein and its activity. If the new proteins exceed this activity they may even find new purposes besides the coordination of metal arrays.

Bibliografie

Ashkenazy, H., O. Penn, A. Doron-Faigenboim, O. Cohen, G. Cannarozzi, O. Zomer, and T. Pupko (2012), “FastML: A web server for probabilistic reconstruction of ancestral sequences.” Nucleic Acids Research, 40, 580–584.

Atici, Ö . and B. Nalbantoglu (2003), “Antifreeze proteins in higher plants.” Phytochemistry, 64, 1187–1196.

Baker, D. (2010), “An exciting but challenging road ahead for computational enzyme design.” Protein Science, 19, 1817–1819.

Bradford, M. M. (1976), “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.” Analytical Biochemistry, 72, 248–254.

Buckley, H. E. (1952), Crystal growth. Wiley, New York. Chao, H., P. L. Davies, and J. F. Carpenter (1996), “Effects of antifreeze proteins on red blood cell survival during cryopreservation.” The Journal of Experimental Biology, 199, 2071–2076.

Chao, H., M. E. Housten, R. S. Hodges, C. M. Kay, B. D. Sykes, M. C. Loewen, P. L. Davies, and F. D. Sönnichsen (1997), “A diminished role for hydrogen bonds in antifreeze protein binding to ice.” Biochemistry, 36, 14652–14660.

Chen, L., A. L. DeVries, and C.-H. C. Cheng (1997a), “Convergent evolution of antifreeze glycoproteins in Antarctic notothenioid fish and Arctic cod.” Proceedings of the National Academy of Sciences of the United States of America, 94, 3817–3822.

Chen, L., A. L. DeVries, and C.-H. C. Cheng (1997b), “Evolution of antifreeze glycoprotein gene from a trypsinogen gene in Antarctic notothenioid fish.” Proceedings of the National Academy of Sciences of the United Stated of America, 94, 3811–3816.

Cheng, C.-H. C. (1998), “Evolution of the diverse antifreeze proteins.” Current Opinion in Genetics and Development, 8, 715–720.

Constans, A. (2005), “Pioneering ionization technique paved the way for proteomics.” The Scientist, 19, 37–41.

Davies, P. L. (2014), “Ice-binding proteins: A remarkable diversity of structures for stopping and starting ice growth.” Trends in Biochemical Sciences, 39, 548–555.

Davies, P. L., J. Baardsnes, M. J. Kuiper, and V. K.Walker (2002), “Structure and function of antifreeze proteins.” Philosophical Transactions of the Royal Society B: Biological Sciences, 357, 927–935.

Davies, P. L. and C. L. Hew (1990), “Biochemistry of fish antifreeze proteins.” The FASEB Journal, 4, 2460–2468.

Davies, P. L., C. L. Hew, and F. L. Fletcher (1988), “Fish antifreeze proteins: Physiology and evolutionary biology.” Canadian Journal of Zoology, 66, 2611–2617.

Delhaise, P., M. Bardiaux, M. De Maeyer, M. Prevost, D. Vanbelle, J. Donneux, I. Lasters, E. Vancustem, P. Alard, and S.Wodak (1988), “The brugel package - toward computeraided-design of macromolecules.” Journal of molecular graphics, 6, 219.

Deng, G., D.W. Andrews, and R. A. Laursen (1997), “Amino acid sequence of a new type of antifreeze protein , from the longhorn sculpin Myoxocephalus octodecimspinosis.” Federation of European Biochemical Societies Letters, 402, 17–20.

Deng, G. and R. A.” Laursen (1998), “Isolation and characterization of an antifreeze protein from the longhorn sculpin, Myoxocephalus octodecimspinosis.” Biochimica et Biophysica Acta, 1388, 305–314.

DeVries, A. L. (1984), “Role of glycopeptides and peptides in inhibition of crystallization of water in polar fishes.” Philosophical Transactions of the Royal Society of London, 304, 575–588.

DeVries, A. L., S. K. Komatsu, and R. E. Feeney (1970), “Chemical and physical properties of freezing point-depressing glycoproteins from Antarctic fishes.” Journal of Biological Chemistry, 245, 2901–2908.

DeVries, A. L. and Y. Lin (1977), “Structure of a peptide antifreeze and mechanism of adsorption to ice.” Biochimica et Biophysica Acta, 495, 388–392.

DeVries, A. L. and D.Wohlschlag (1969), “Freezing resistance in some Antarctic fishes.” Science, 163, 1073–1075.

Doucet, D., M. G. Tyshenko, M. J. Kuiper, S. P. Graether, B. D. Sykes, A. J. Daugulis, P. L. Davies, and V. K. Walker (2000), “Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.” European Journal of Biochemistry, 267, 6082–6088.

Drevin, I. and B.-L. Johansson (1991), “Stability of Superdex 75 prep grade and Superdex200 prep grade under different chromatographic conditions.” Journal of Chromatography, 547, 21–30.

Drori, R., Y. Celik, P. L. Davies, and I. Braslavsky (2014), “Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.” Journal of The Royal Society Interface, 11, 10.

Duman, J. and K. Horwath (1983), “The role of hemolymph proteins in the cold tolerance of insects.” Annual review of physiology, 45, 261–270.

Duman, J. G. (1979), “Subzero temperature tolerance in spiders: The role of thermal hysteresis factors.” Journal of Comparative Physiology, 131, 347–352.

Duman, J. G. (2001), “Antifreeze and ice nucleator proteins in terrestrial arthropods.” Annual Review of Physiology, 63, 327–357.

Duman, J. G. and A. L. DeVries (1976), “Isolation, characterization and physical properties of protien antifreezes from the winter flounder, Pseudopleuronectes americanus.” Comparative Biochemistry and Physiology, 533, 375–380.

Duman, J. G. and T. M. Olsen (1993), “Thermal hysteresis protein activity in bacteria, fungi, and phylogenetically diverse plants.” Cryobiology, 30, 322–328.

Duman, J. H., J. L. Patterson, J. J. Kozak, and A. L. DeVries (1980), “Isopiestic determination of water binding by fish antifreeze glycoproteins.” Biochimica et Biophysica Acta, 626, 332–336.

Emmert-Streib, F. (2012), “Limitation of gene duplication models: Evolution of modules in protein interaction networks.” Public Library of Science One, 7, 1–13.

Farewell, A., K. Kvint, and T. Nyström (1998), “uspB, a new _S-regulated gene in Escherichia coli which is required for stationary-phase resistance to ethanol.” Journal of Bacteriology, 180, 6140–6147.

Fleishman, S. J., J. E. Corn, E.-M. Strauch, T. A. Whitehead, J. Karanicolas, and D. Baker (2011), “Hotspot-centric De Novo design of protein binders.” Journal of Molecular Biology, 413, 1047–1062.

Franks, F. (1982), The properties of aqueous solutions at subzero temperatures. Plenum Press, New York. In Water, volume 7: Water and Aqueous Solutions at Subzero Temperatures.

Gagne, M., L. Spyracopoulos, S. P. Graether, Z. Jia, P. L. Davies, and B. D. Sykes (2003), “Spruce budworm antifreeze protein: Changes in structure and dynamics at low temperature.” Journal of Molecular Biology, 327, 1155–1168.

Garnham, C. P., R. L. Campbell, and P. L. Davies (2011), “Anchored clathrate waters bind antifreeze proteins to ice.” Proceedings of the National Academy of Sciences, 108, 7363–7367.

Gauthier, S. Y., A. J. Scotter, F.-H. Lin, J. Baardsnes, G. L. Fletcher, and P. L. Davies (2008), “A re-evaluation of the role of type IV antifreeze protein.” Cryobiology, 57, 292–296.

Gilbert, J. A., P. L. Davies, and J. Laybourn-parry (2005), “A hyperactive , Ca2+-dependent antifreeze protein in an Antarctic bacterium.” FEMS Microbiology Letters, 245, 67–72.

Gille, C. and C. Frömmel (2001), “STRAP: editor for STRuctural Alignments of Proteins.” Bioinformatics, 17, 377–378.

Graham, L. A., Y-C. Liou, V. K.Walker, and P. L. Davies (1997), “Hyperactive antifreeze protein from beetles.” Nature, 388, 727–728.

Griffith, M. and K. V. Ewart (1995), “Antifreeze proteins and their potential use in frozen foods.” Biotechnology advances, 13, 375–402.

Guilhaus, M. (1995), “Principles and instrumentation in time-of-flight mass spectrometry.” Journal of Mass Spectrometry, 30, 1519–1532.

Hakim, A., J. B. Nguyen, K. Basu, D. F. Zhu, D. Thakral, P. L. Davies, F. J. Isaacs, Y. Modis, and W. Meng (2013), “Crystal structure of an insect antifreeze protein and its implications for ice binding.” The Journal of Biological Chemistry, 288, 12295–12304.

Harding, M. M., P. I. Anderberg, and A. D. J. Haymet (2003), “Antifreeze glycoproteins from polar fish.” European Journal of Biochemistry, 270, 1381–1392.

Haschemeyer, A. E. V., W. Guschlbaur, and A. L. DeVries (1977), “Water binding by antifreeze glycoproteins from Antarctic fish.” Nature, 269, 87–88.

Haymet, A. D. J., L. G. Ward, and M. M. Harding (1999), “Winter flounder “antifreeze” proteins: Synthesis and ice grotwh inhibition of analogues that probe the relative importance of hydrophobic and hydrogen-bonding interactions.” Journal of the American Chemical Society, 121, 941–948.

Haymet, A. D. J., L. G.Ward, and M. M. Harding (2001), “Hydrophobic analogues of the winter flounder antifreeze protein.” Federation of European Biochemical Societies letters, 491, 285–288.

Haymet, A. D. J., L. G. Ward, M. M. Harding, and C. A. Knight (1998), “Valine substituted winter flounder antifreeze: Preservation of ice growth hysteresis.” Federation of European Biochemical Societies letters, 430, 301–306.

Hobbs, R. S., M. A. Shears, L. A. Graham, P. L. Davies, and G. L. Fletcher (2011), “Isolation and characterization of type I antifreeze proteins from cunner, Tautogolabrus adspersus, order Perciformes.” Federation of European Biochemical Societies, 278, 3699–3710.

Hyzak, L., R. Moos, F. von Rath, V. Wulf, M. Wirtz, D. Melchior, H.-W. Kling, M. Köhler, S. Gäb, and O. J. Schmitz (2011), “Quantitative matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis of synthetic polymers and peptides.” Analytical Chemistry, 83, 9467–9471.

Isgro, T. A., M. Sotomayor, and E. Cruz-Chu (2014), Case study: Water and ice. University of Illinois.

Karim, O. A. and A. D. J. Haymet (1988), “The ice/water interface: A molecular dynamics simulation study.” The Journal of Chemical Physics, 89, 6889–6896.

Kaufmann, K. W., G. H. Lemmon, S. L. DeLuca, J. H. Sheehan, and J. Meiler (2010), “Practically useful: What the Rosetta Protein Modelling Suite can do for you.” Biochemistry, 49, 2987–2998.

Knight, C. A. (1967), The freezing of supercooled liquids. D. Van Nostrand Company, Inc., New Jersey.

Knight, C. A., E. Driggers, and A. L. DeVries (1993), “Adsorption to ice of fish antifreeze glycopeptides 7 and 8.” Biophysical Journal, 64, 252–259.

Kondo, H., Y. Hanada, H. Sugimoto, T. Hoshino, C. P. Garnham, P. L. Davies, and S. Tsuda (2012), “Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.” Proceedings of the National Academy of Sciences, 109, 9360–9365.

Koushafar, H., L. Pham, C. Lee, and B. Rubinsky (1997), “Chemical adjuvant cryosurgery with antifreeze proteins.” Journal of Surgical Oncology, 66, 114–121.

Lauersen, K. J., A. Brown, A. Middleton, P. L. Davies, and V. K.Walker (2011), “Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne.” Crybiology, 62, 194–201.

Leinala, E. K., P. L. Davies, D. Doucet, M. G. Tyshenko, V. K.Walker, and Z. Jia (2002a), “A _-helical antifreeze protein isoform with increased activity.” The Journal of Biological Chemistry, 277, 33349–33352.

Leinala, E. K., P. L. Davies, and Z. Jia (2002b), “Crystal structure of _-helical antifreeze protein points to a general ice binding model.” Structure, 10, 619–627.

Li, C. and C. Jin (2004), “Letters to the Editor: 1H, 13C and 15N resonance assignments of the antifreeze protein cfAFP-501 from spruce budworm at different temperatures.” Journal of Biomolecular NMR, 30, 101–102.

Liou, Y.-C., A. Tocilj, P. L. Davies, and Z. Jia (2000), “Mimicry of ice structure by surface hydroxyls and water of a _-helix antifreeze protein.” Nature, 406, 322–324.

Liu, Y., Z. Li, Q. Lin, J. Kosinski, J. Seetharaman, J. M. Bujnicki, Sivaraman J., and C.-L. Hew (2007), “Structure and evolutionary origin of calcium dependent herring type II antifreeze protein.” Public Library of Science ONE, 2, 1–11.

Louis-Jeune, C., M. A. Andrade-Navarro, and C. Perez-Iratxeta (2011), “Prediction of protein secondary structure from circular dichroism using theoretically derived spectra.” Proteins: Structure, Function, and Bioinformatics, 80, 374–381.

Middleton, A. J., A. M. Brown, P. L. Davies, and V. K. Walker (2009), “Identification of the ice-binding face of a plant antifreeze protein.” Federation of European Biochemical Societies Letters, 583, 815–819.

Middleton, A. J., C. B. Marshall, F. Faucher, M. Bar-dolev, I. Braslavsky, R. L. Campbell, V. K. Walker, and P. L. Davies (2012), “Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.” Journal of Molecular Biology, 416, 713–724.

Mok, Y.-F., F.-H. Lin, L. A. Graham, Y. Celik, I. Braslavsky, and P. L. Davies (2010), “Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.” Biochemistry, 49, 2593–2603.

Muldrew, K., J. Rewcastle, B. J. Donnelly, J. C. Saliken, S. Liang, S. Goldie, M. Olson, R. Baissalov, and G. Sandison (2001), “Flounder antifreeze peptides increase the efficacy of cryosurgery.” Cryobiology, 42, 182–189.

Nishimiya, Y., H. Kondo, M. Takamichi, H. Sugimoto, M. Suzuki, A. Miura, and S. Tsuda (2008), “Crystal structure and mutational analysis of calcium-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus.” Journal of Molecular Biology, 382, 734–746.

Novagen (2003), pET system manual, 10 edition.

Novagen (n.d.), “Competent cells: What a difference a strain makes.” Merck KGaA, Darmstadt, Germany.

Ochlal, E.-L. (1991), “Biomineralization principle.” Principles and Applications in Bioinorganic Chemistry, 68, 627–630.

Perez-Iratxeta, C. and M. A. Andrade-Navarro (2008), “K2D2: Estimation of protein secondary structure from circular dichroism spectra.” BioMed Central Structural Biology, 8, 1–5.

Pupko, T., I. Pe’er, M. Hasegawa, D. Graur, and N. Friedman (2002), “A branch-andbound algorithm for the for the inference of ancestral amino-acids sequences when the replacement rate varies among sites: Application to the evolution of five gene families.” Bioinformatics, 18, 1116–1123.

Pupko, T., I. Pe’er, R. Shamir, and D. Graur (2000), “A fast algorithm for join reconstruction of ancestral amino acids sequences.” Molecular Biology and Evolution, 17, 890–896.

Rath, A., M. Glibowicka, V. G. Nadeau, G. Chen, and C. M. Deber (2009), “Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.” Proceedings of the Natoinal Academy of Sciences, 106, 1760–1765.

Raymond, J. A. (1976), Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Ph.D. thesis, University of California.

Raymond, J. A. (2000), “Distribution and partial characterization of ice-active molecules associated with sea-ice diatoms.” Polar Biology, 23, 721–729.

Raymond, J. A. and A. L. DeVries (1977), “Adsorption inhibition as a mechanism of freezing resistance in polar fishes.” Proceedings of the National Academy of Sciences of the United States of America, 74, 2589–93.

Rosano, G. L. and E. A. Ceccarelli (2014), “Recombinant protein expression in Escherichia coli: Advances and challenges.” Frontiers in Microbiology, 5, 1–17.

Sanders, C. J. (1991), Biology of North American spruce budworms. Elsevlier, Amsterdam. In tortricid pests, Their biology, natural enemies and control, volume 7: Tortricids in forestry.

Scheraga, G. A., G. Nemethy, and I. Z. Steinberg (1962), “The contribution of hydrophobic bonds to the thermal stability of protein conformations.” Journal of Biological Chemistry, 237, 2506–2508.

Schrödinger, LLC (2015), “The PyMOL molecular graphics system, version 1.8.” KEY: PyMOL, ANNOTATION: PyMOL The PyMOL Molecular Graphics System, Version 1.8, Schrödinger, LLC.

Scotter, A. J., C. B. Marshall, L. A. Graham, J. A. Gilbert, C. P. Garnham, and P. L. Davies (2006), “The basis for hyperactivity of antifreeze proteins.” Cryobiology, 53, 229–239.

Shier, W. T., Y. Lin, and A. L. DeVries (1972), “Structure and mode of action of glycoproteins from an Antarctic fish.” Biochimica et Biophysica Acta, 263, 406–413.

Sönnichsen, F., B. Sykes, H. Chao, and P. L. Davies (1993), “The nonhelical structure of antifreeze protein type III.” Science, 259, 1154–1157.

Sun, T., F.-H. Lin, R. L. Campbell, J. S. Allingham, and P. L. Davies (2014), “An antifreeze proteins folds with an interior network of more than 400 semi-clathrate waters.” Science, 343, 795–798.

Tablin, F., A. E. Oliver, N. J. Walker, L. M. Crowe, and J. H. Crowe (1996), “Membrane phase transition of intact human platelets: Correlation with cold-induced activation.” Journal of Cellular Physiology, 168, 305–313.

Taylor, R. G., D. C. Walker, and R. Mclnnes (1993), “E.coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing.” Nucleic Acids Research, 21, 1677–1678.

Teeter, M. M. (1984), “Water structure of a hydrophobic protein at atomic resolution: Pentagon rings of water molecules in crystals of crambin.” Proceedings of the National Academy of Sciences, 81, 6014–6018.

Tursman, D., J. G. Duman, and C. A. Knight (1994), “Freeze tolerance adaptations in the centipede Lithobius forficatus.” Journal of Experimental Zoology, 268, 347–353.

Tyshenko, M. G., D. Doucet, P. L. Davies, and V. K. Walker (1997), “The antifreeze potential of the spruce budworm thermal hysteresis protein.” Nature Biotechnology, 15, 887–890.

Voet, A. R. D., H. Noguchi, C. Addy, D. Simoncini, D. Terada, S. Unzai, S.-Y. Park, K. Y. J. Zhang, and J. R. H. Tame (2014), “Computational design of a self-assembling symmetrical _-propeller protein.” Proceedings of the National Academy of Sciences, 111, 15102–15107.

Voet, A. R. D., H. Noguchi, C. Addy, K. Y. J. Zhang, and J. R. H. Tame (2015), “Biomineralization of a cadmium chloride nanocrystal by a designed symmetrical protein.” Angewandte Chemie International Edition, 54, 9857–9860.

Wang, T., Q. Zhu, X. Yang, J. R. Layne, and A. L. DeVries (1994), “Antifreeze glycoproteins from Antarctic Notothenioid fishes fail to protect the rat cardiac explant during hypothermic and freezing preservation.” Cryobiology, 31., 185–192.

Woody, R. W. (1995), “Circular dichroism.” Methods in Enzymology, 246, 34–71.

Wu, Y., J. Banoub, S. V. Goddard, M. H. Kao, and G. L. Fletcher (2001), “Antifreeze glycoproteins: relationship between molecular weight, thermal hysteresis and the inhibition of leakage from liposomes during thermotropic phase transition.” Comparative Biochemistry and Physiology Part B, 128, 265–273.

Yeh, Y. and R. E. Feeney (1996), “Antifreeze proteins : Structures and mechanisms of function.” Chemical Reviews, 96.

Yu, S. O. W. (2010), Antifreeze proteins: Activity comparisons and de novo design of an ice-binding protein. Master’s thesis, Queen’s University, Kingstone, Canada.

Zhang, W. and R. A. Laursen (1998), “Structure-function relationships in a type I antifreeze polypeptide. The role of threonine methyl and hydroxyl groups in antifreeze activity.” The Journal of Biological Chemistry, 273, 34806–34812.

Zhang, z., S. Schwartz, L. Wagner, and W. Miller (2000), “A greedy algorithm for aligning DNA sequences.” Journal of Computation Biology, 7, 203–214.

Download scriptie (12.11 MB)
Universiteit of Hogeschool
KU Leuven
Thesis jaar
2016
Promotor(en)
Arnout Voet