Abstract
Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.
Similar content being viewed by others
References
Adamczak R, Porollo A, Meller J (2005) Combining prediction of secondary structure and solvent accessibility in proteins. Proteins Struct Funct Bioinform 59:467–475
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Amir G, Rubinsky B, Kassif Y, Horowitz L, Smolinsky AK, Lavee J (2003) Preservation of myocyte structure and mitochondrial integrity in subzero cryopreservation of mammalian hearts for transplantation using antifreeze proteins: an electron microscopy study. Eur J Cardiothorac Surg 24:292–297
Barrett J (2001) Thermal hysteresis proteins. Int J Biochem Cell Biol 33:105–117
Bayer-Giraldi M, Uhlig C, John U, Mock T, Valentin K (2010) Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis. Environ Microbiol 12:1041–1052
Biegert A, Mayer C, Remmert M, Söding J, Lupas AN (2006) The MPI bioinformatics toolkit for protein sequence analysis. Nucleic Acids Res 34:W335–W339
Boo S, Wong C, Rodrigues K, Najimudin N, Murad A, Mahadi N (2013) Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR. Polar Biol 36:381–389
Bowie J, Luthy R, Eisenberg D (1991) A method to identify protein sequences that fold into a known three-dimensional structure. Science 253:164–170
Bravo LA, Griffith M (2005) Characterization of antifreeze activity in Antarctic plants. J Exp Bot 56:1189–1196
Christner B (2010) Bioprospecting for microbial products that affect ice crystal formation and growth. Appl Microbiol Biotechnol 85:481–489
Colovos C, Yeates TO (1993) Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 2:1511–1519
Davies PL, Baardsnes J, Kuiper MJ, Walker VK (2002) Structure and function of antifreeze proteins. Philos Trans R Soc Lond 357:927–935
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797
Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, Pieper U, Sali A (2001) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics 15:5.6.1–5.6.30
Felsenstein J (1996) Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods. In: Russell FD (ed) Methods in enzymology. Academic Press, pp 418–27
Fletcher GL, Hew CL, Davies PL (2001) Antifreeze proteins of teleost fishes. Annu Rev Physiol 63:359–390
Garnham CP, Gilbert JA, Hartman CP, Campbell RL, Laybourn-Parry J, Davies PL (2008) A Ca2+-dependent bacterial antifreeze protein domain has a novel beta-helical ice-binding fold. Biochem J 411:171–180
Garnham CP, Campbell RL, Davies PL (2011) Anchored clathrate waters bind antifreeze proteins to ice. Proc Natl Acad Sci USA 108:7363–7367
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel D, Bioroch A (2005) Protein identification and analysis tools on the ExPASy Server. In: Walker JM (ed) The proteomics protocol handbook. Humana Press, Totowa, pp 571–607
Graether SP, Kuiper MJ, Gagné SM, Walker VK, Jia Z, Sykes BD, Davies PL (2000) Beta-helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect. Nature 406:325–328
Griffith M, Ewart KV (1995) Antifreeze proteins and their potential use in frozen foods. Biotechnol Adv 13:375–402
Griffith M, Yaish MWF (2004) Antifreeze proteins in overwintering plants: a tale of two activities. Trends Plant Sci 9:399–405
Hashim N, Bharudin I, Nguong D, Higa S, Bakar F, Nathan S, Rabu A, Kawahara H, Illias R, Najimudin N, Mahadi N, Murad A (2013) Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12. Extremophiles 17:63–73
Hoshino T, Kiriaki M, Ohgiya S, Fujiwara M, Kondo H, Nishimiya Y, Yumoto I, Tsuda S (2003) Antifreeze proteins from snow mold fungi. Can J Bot 81:1175–1181
Jia Z, Davies PL (2002) Antifreeze proteins: an unusual receptor–ligand interaction. Trends Biochem Sci 27:101–106
Kawahara H, Iwanaka Y, Higa S, Muryoi N, Sato M, Honda M, Omura H, Obata H (2007) A novel, intracellular antifreeze protein in an antarctic bacterium, Flavobacterium xanthum. Cryoletters 28:39–49
Kim H, Lee J, Do H, Jung W (2014) Production of antifreeze proteins by cold-adapted yeasts. In: Buzzini P, Margesin R (eds) Cold-adapted yeasts. Springer, Heidelberg, pp 259–280
Knight CA, Driggers E, DeVries AL (1993) Adsorption to ice of fish antifreeze glycopeptides 7 and 8. Biophys J 64:252–259
Kondo H, Hanada Y, Sugimoto H, Hoshino T, Garnham CP, Davies PL, Tsuda S (2012) Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation. Proc Natl Acad Sci USA 109:9360–9365
Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291
Lee JK, Park KS, Park S, Park H, Song YH, Kang S-H, Kim HJ (2010) An extracellular ice-binding glycoprotein from an Arctic psychrophilic yeast. Cryobiology 60:222–228
Lee JH, Park AK, Do H, Park KS, Moh SH, Chi YM, Kim HJ (2012) Structural basis for antifreeze activity of ice-binding protein from Arctic yeast. J Biol Chem 287:11460–11468
Melo F, Sali A (2007) Fold assessment for comparative protein structure modeling. Protein Sci 16:2412–2426
Muryoi N, Sato M, Kaneko S, Kawahara H, Obata H, Yaish MWF, Griffith M, Glick BR (2004) Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2. J Bacteriol 186:5661–5671
Park KS, Do H, Lee JH, Park SI, Kim EJ, Kim S-J, Kang S-H, Kim HJ (2012) Characterization of the ice-binding protein from Arctic yeast Leucosporidium sp. AY30. Cryobiology 64:286–296
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786
Ramli A, Mahadi N, Shamsir M, Rabu A, Joyce-Tan K, Murad A, Illias R (2012) Structural prediction of a novel chitinase from the psychrophilic Glaciozyma antarctica PI12 and an analysis of its structural properties and function. J Comput Aided Mol Des 26:947–961
Raymond JA, DeVries AL (1977) Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci USA 74:2589–2593
Raymond JA, Kim HJ (2012) Possible role of horizontal gene transfer in the colonization of sea ice by algae. PLoS One 7:e35968
Regand A, Goff HD (2006) Ice recrystallization inhibition in ice cream as affected by ice structuring proteins from winter wheat grass. J Dairy Sci 89:49–57
Robinson CH (2001) Cold adaptation in Arctic and Antarctic fungi. New Phytol 151:341–353
Sharp KA (2011) A peek at ice binding by antifreeze proteins. Proc Natl Acad Sci USA 108:7281–7282
Sicheri F, Yang DSC (1995) Ice-binding structure and mechanism of an antifreeze protein from winter flounder. Nature 375:427–431
Sönnichsen FD, DeLuca CI, Davies PL, Sykes BD (1996) Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein ice interaction. Structure 4:1325–1337
Sun X, Griffith M, Pasternak JJ, Glick BR (1995) Low temperature growth, freezing survival, and production of antifreeze protein by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Can J Microbiol 41:776–784
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) Software Version 4.0. Mol Biol Evol 24:1596–1599
Tsvetkova NM, Phillips BL, Krishnan VV, Feeney RE, Fink WH, Crowe JH, Risbud SH, Tablin F, Yeh Y (2002) Dynamics of antifreeze glycoproteins in the presence of ice. Biophys J J82:464–473
Uhlig C, Kabisch J, Palm GJ, Valentin K, Schweder T, Krell A (2011) Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae). Cryobiology 63:220–228
Venketesh S, Dayananda C (2008) Properties, potentials, and prospects of antifreeze proteins. Crit Rev Biotechnol 28:57–82
Wu DW, Duman JG, Cheng C-HC, Castellino FJ (1991) Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis. J Comp Phys B 161:271–278
Xiao N, Suzuki K, Nishimiya Y, Kondo H, Miura A, Tsuda S, Hoshino T (2010) Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis. FEBS J 277:394–403
Zhang C, Zhang H, Wang L (2007) Effect of carrot (Daucus carota) antifreeze proteins on the fermentation capacity of frozen dough. Food Res Int 40:763–769
Acknowledgments
This research was funded by the Ministry of Science, Technology and Innovation (MOSTI), Malaysia, under the research grants 10-05-16-MB002 and 07-05-MGI-GMB014. We thank Professor William J. Broughton for critical reading the manuscript and helpful comments. We acknowledge support given by the Australian Antarctic Division and the Malaysian Antarctic Research Programme (MARP) of the Academy of Science, Malaysia.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hashim, N.H.F., Sulaiman, S., Abu Bakar, F.D. et al. Molecular cloning, expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica . Polar Biol 37, 1495–1505 (2014). https://doi.org/10.1007/s00300-014-1539-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00300-014-1539-1