Amino Acids

, Volume 46, Issue 11, pp 2615–2625 | Cite as

The functional role of Cys3–Cys4 loop in hydrophobin HGFI

  • Baolong Niu
  • Yanbo Gong
  • Xianghua Gao
  • Haijin Xu
  • Mingqiang Qiao
  • Wenfeng LiEmail author
Original Article


Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic–hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3–Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3–Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water–air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the Congo Red (CR) absorption spectra shift (after the THT(CR)/HGFI-AR mixed aqueous solution was drastically vortexed). Remarkably, replacement of the Cys3–Cys4 loop could impair the rodlet formation of the class I hydrophobin HGFI. So, it could be speculated that the Cys3–Cys4 loop plays an important role in conformation and functionality, when the class I hydrophobin HGFI self-assembles at hydrophobic–hydrophilic interfaces.


Hydrophobin HGFI Self-assembly Cys3–Cys4 loop Rodlets structure 



This research was financially supported by the National Natural Science Foundation of China (31170066), Tianjin Key Research Program of Application Foundation and Advanced Technology (12JCZDJC22600) and the Natural Science Foundation of Shanxi Province (2014021020-3).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Askolin S, Linder M, Scholtmeijer K, Tenkanen M, Penttilä M, de Vocht ML, Wösten HAB (2006) Interaction and comparison of a class I hydrophobin from Schizophyllum commune and class II hydrophobins from Trichoderma reesei. Biomacromolecules 7(4):1295–1301. doi: 10.1021/bm050676s PubMedCrossRefGoogle Scholar
  2. Basheva ES, Kralchevsky PA, Danov KD, Stoyanov SD, Blijdenstein TBJ, Pelan EG, Lips A (2011) Self-assembled bilayers from the protein HFBII hydrophobin: nature of the adhesion energy. Langmuir 27(8):4481–4488. doi: 10.1021/la2001943 PubMedCrossRefGoogle Scholar
  3. Burrowes O-J, Diamond G, Lee T-C (2005) Recombinant expression of pleurocidin cDNA using the Pichia pastoris expression system. J Biomed Biotechnol 4:374–384. doi: 10.1155/jbb.2005.374 CrossRefGoogle Scholar
  4. Butko P, Buford JP, Goodwin JS, Stroud PA, McCormick CL, Cannon GC (2001) Spectroscopic evidence for amyloid-like interfacial self-assembly of hydrophobin Sc3. Biochem Biophys Res Commun 280(1):212–215. doi: 10.1006/bbrc.2000.4098
  5. de Vocht ML, Scholtmeijer K, van der Vegte EW, de Vries OMH, Sonveaux N, Wösten HAB, Ruysschaert J-M, Hadziioannou G, Wessels JGH, Robillard GT (1998) Structural characterization of the hydrophobin SC3, as a monomer and after self-assembly at hydrophobic/hydrophilic interfaces. Biophys J 74(4):2059–2068. doi: 10.1016/S0006-3495(98)77912-3
  6. De Vocht ML, Reviakine I, Ulrich W-P, Bergsma-Schutter W, Wösten HAB, Vogel H, Brisson A, Wessels JGH, Robillard GT (2002) Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates. Protein Sci 11(5):1199–1205. doi: 10.1110/ps.4540102 PubMedCrossRefPubMedCentralGoogle Scholar
  7. de Vries OMH, Moore S, Arntz C, Wessels JGH, Tudzynski P (1999) Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. Eur J Biochem 262(2):377–385. doi: 10.1046/j.1432-1327.1999.00387.x PubMedCrossRefGoogle Scholar
  8. Hakanpää J, Paananen A, Askolin S, Nakari-Setälä T, Parkkinen T, Penttilä M, Linder MB, Rouvinen J (2004) Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. J Biol Chem 279(1):534–539. doi: 10.1074/jbc.M309650200 PubMedCrossRefGoogle Scholar
  9. Hakanpää J, Linder M, Popov A, Schmidt A, Rouvinen J (2006) Hydrophobin HFBII in detail: ultrahigh-resolution structure at 0.75 A. Acta Crystallogr Sect D 62(4):356–367. doi: 10.1107/S0907444906000862
  10. Hou S, Li X, Li X, Feng X-Z, Wang R, Wang C, Yu L, Qiao M-Q (2009) Surface modification using a novel type I hydrophobin HGFI. Anal Bioanal Chem 394(3):783–789. doi: 10.1007/s00216-009-2776-y PubMedCrossRefGoogle Scholar
  11. Houmadi S, Ciuchi F, De Santo MP, De Stefano L, Rea I, Giardina P, Armenante A, Lacaze E, Giocondo M (2008) Langmuir–Blodgett film of hydrophobin protein from Pleurotus ostreatus at the air–water interface. Langmuir 24(22):12953–12957. doi: 10.1021/la802306r PubMedCrossRefGoogle Scholar
  12. Janssen MI, Leeuwen MBMv, Kooten TGv, Vries Jd, Dijkhuizen L, Wösten HAB (2004) Promotion of fibroblast activity by coating with hydrophobins in the β-sheet end state. Biomaterials 25(14):2731–2739. doi: 10.1016/j.biomaterials.2003.09.060 PubMedCrossRefGoogle Scholar
  13. Kershaw MJ, Talbot NJ (1998) Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis. Fungal Genet Biol 23(1):18–33. doi: 10.1006/fgbi.1997.1022
  14. Klunk WE, Jacob RF, Mason RP (1999) Quantifying amyloid by congo red spectral shift assay. In: Ronald W (ed) Methods in enzymology, vol 309. Academic Press, New York, pp 285–305. doi: 10.1016/S0076-6879(99)09021-7
  15. Kwan AHY, Winefield RD, Sunde M, Matthews JM, Haverkamp RG, Templeton MD, Mackay JP (2006) Structural basis for rodlet assembly in fungal hydrophobins. Proc Natl Acad Sci USA 103(10):3621–3626. doi: 10.1073/pnas.0505704103 PubMedCrossRefPubMedCentralGoogle Scholar
  16. Kwan AH, Macindoe I, Vukašin PV, Morris VK, Kass I, Gupte R, Mark AE, Templeton MD, Mackay JP, Sunde M (2008) The Cys3–Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity. J Mol Biol 382(3):708–720. doi: 10.1016/j.jmb.2008.07.034
  17. LeVine Iii H (1999) Quantification of β-sheet amyloid fibril structures with thioflavin T. In: Ronald W (ed) Methods in enzymology, vol 309. Academic Press, New York, pp 274–284. doi: 10.1016/S0076-6879(99)09020-5
  18. Linder MB (2009) Hydrophobins: proteins that self assemble at interfaces. Curr Opin Colloid Interf Sci 14(5):356–363. doi: 10.1016/j.cocis.2009.04.001
  19. Linder MB, Szilvay GR, Nakari-Setälä T, Penttilä ME (2005) Hydrophobins: the protein-amphiphiles of filamentous fungi. FEMS Microbiol Rev 29(5):877–896. doi: 10.1016/j.femsre.2005.01.004 PubMedCrossRefGoogle Scholar
  20. Lugones LG, Wösten HAB, Wessels JGH (1998) A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom). Microbiology 144(8):2345–2353. doi: 10.1099/00221287-144-8-2345 PubMedCrossRefGoogle Scholar
  21. Ma H, Snook LA, Tian C, Kaminskyj SGW, Dahms TES (2006) Fungal surface remodelling visualized by atomic force microscopy. Mycol Res 110(8):879–886. doi: 10.1016/j.mycres.2006.06.010
  22. Ma A, Shan L, Wang H, Du Z, Xie B (2008) Partial characterization of a hydrophobin protein Po.HYD1 purified from the oyster mushroom Pleurotus ostreatus. World J Microbiol Biotechnol 24(4):501–507. doi: 10.1007/s11274-007-9500-x CrossRefGoogle Scholar
  23. Macindoe I, Kwan AH, Ren Q, Morris VK, Yang W, Mackay JP, Sunde M (2012) Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS. Proc Natl Acad Sci USA. doi: 10.1073/pnas.1114052109 PubMedPubMedCentralGoogle Scholar
  24. Morris VK, Ren Q, Macindoe I, Kwan AH, Byrne N, Sunde M (2011) Recruitment of class I hydrophobins to the air:water interface initiates a multi-step process of functional amyloid formation. J Biol Chem 286(18):15955–15963. doi: 10.1074/jbc.M110.214197 PubMedCrossRefPubMedCentralGoogle Scholar
  25. Niu B, Huang Y, Zhang S, Wang D, Xu H, Kong D, Qiao M (2012a) Expression and characterization of hydrophobin HGFI fused with the cell-specific peptide TPS in Pichia pastoris. Protein Express Purif 83(1):92–97. doi: 10.1016/j.pep.2012.03.004
  26. Niu B, Wang D, Yang Y, Xu H, Qiao M (2012b) Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry. Amino Acids 43(2):763–771. doi: 10.1007/s00726-011-1126-5 PubMedCrossRefGoogle Scholar
  27. Paananen A, Vuorimaa E, Torkkeli M, Penttilä M, Kauranen M, Ikkala O, Lemmetyinen H, Serimaa R, Linder MB (2003) Structural hierarchy in molecular films of two class II hydrophobins. Biochemistry 42(18):5253–5258. doi: 10.1021/bi034031t PubMedCrossRefGoogle Scholar
  28. Paslay LC, Falgout L, Savin DA, Heinhorst S, Cannon GC, Morgan SE (2013) Kinetics and control of self-assembly of ABH1 hydrophobin from the edible White Button mushroom. Biomacromolecules 14(7):2283–2293. doi: 10.1021/bm400407c PubMedCrossRefGoogle Scholar
  29. Sbrana F, Fanelli D, Vassalli M, Carresi L, Scala A, Pazzagli L, Cappugi G, Tiribilli B (2010) Progressive pearl necklace collapse mechanism for cerato-ulmin aggregation film. Eur Biophys J 39(6):971–977. doi: 10.1007/s00249-009-0465-6 PubMedCrossRefGoogle Scholar
  30. Scholtmeijer K, Wessels J, Wösten H (2001) Fungal hydrophobins in medical and technical applications. Appl Microbiol Biotechnol 56(1–2):1–8. doi: 10.1007/s002530100632 PubMedCrossRefGoogle Scholar
  31. Serimaa R, Torkkeli M, Paananen A, Linder M, Kisko K, Knaapila M, Ikkala O, Vuorimaa E, Lemmetyinen H, Seeck O (2003) Self-assembled structures of hydrophobins HFBI and HFBII. J Appl Crystallogr 36(3 Part 1):499–502. doi:doi: 10.1107/S0021889803000578
  32. Stroud PA, Goodwin JS, Butko P, Cannon GC, McCormick CL (2003) Experimental evidence for multiple assembled states of Sc3 from Schizophyllum commune. Biomacromolecules 4(4):956–967. doi: 10.1021/bm034045e PubMedCrossRefGoogle Scholar
  33. Sunde M, Kwan AHY, Templeton MD, Beever RE, Mackay JP (2008) Structural analysis of hydrophobins. Micron 39(7):773–784. doi: 10.1016/j.micron.2007.08.003
  34. Talbot NJ, Kershaw MJ, Wakley GE, De Vries O, Wessels J, Hamer JE (1996) MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell Online 8(6):985–999. doi: 10.1105/tpc.8.6.985 CrossRefGoogle Scholar
  35. Trembley ML, Ringli C, Honegger R (2002) Hydrophobins DGH1, DGH2, and DGH3 in the lichen-forming basidiomycete Dictyonema glabratum. Fungal Genet Biol 35(3):247–259. doi: 10.1006/fgbi.2001.1325
  36. Wang X, Permentier HP, Rink R, Kruijtzer JAW, Liskamp RMJ, Wösten HAB, Poolman B, Robillard GT (2004) Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry. Biophys J 87(3):1919–1928PubMedCrossRefPubMedCentralGoogle Scholar
  37. Wang Z, Feng S, Huang Y, Li S, Xu H, Zhang X, Bai Y, Qiao M (2010a) Expression and characterization of a Grifola frondosa hydrophobin in Pichia pastoris. Protein Express Purif 72(1):19–25. doi: 10.1016/j.pep.2010.03.017
  38. Wang Z, Huang Y, Li S, Xu H, Linder MB, Qiao M (2010b) Hydrophilic modification of polystyrene with hydrophobin for time-resolved immunofluorometric assay. Biosensors Bioelectron 26(3):1074–1079. doi: 10.1016/j.bios.2010.08.059
  39. Wessels JGH (1994) Developmental regulation of fungal cell wall formation. Annu Rev Phytopathol 32(1):413–437. doi: 10.1146/ CrossRefGoogle Scholar
  40. Wessels JGH (1996) Hydrophobins: proteins that change the nature of the fungal surface. Adv Microb Physiol 38. doi: 10.1016/s0065-2911(08)60154-x
  41. Wessels J, De Vries O, Asgeirsdottir SA, Schuren F (1991) Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum. Plant Cell Online 3(8):793–799. doi: 10.1105/tpc.3.8.793 CrossRefGoogle Scholar
  42. Wösten HAB (2001) Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55(1):625–646. doi: 10.1146/annurev.micro.55.1.625 PubMedCrossRefGoogle Scholar
  43. Wösten HAB, de Vocht ML (2000) Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta Rev Biomembr 1469(2):79–86. doi: 10.1016/S0304-4157(00)00002-2
  44. Wösten HAB, Wessels JGH (1997) Hydrophobins, from molecular structure to multiple functions in fungal development. Mycoscience 38(3):363–374. doi: 10.1007/bf02464099 CrossRefGoogle Scholar
  45. Wösten HAB, van Wetter M-A, Lugones LG, van der Mei HC, Busscher HJ, Wessels JGH (1999) How a fungus escapes the water to grow into the air. Curr Biol 9(2):85–88. doi: 10.1016/S0960-9822(99)80019-0
  46. Yang W, Ren Q, Wu Y-N, Morris VK, Rey AA, Braet F, Kwan AH, Sunde M (2013) Surface functionalization of carbon nanomaterials by self-assembling hydrophobin proteins. Biopolymers 99(1):84–94. doi: 10.1002/bip.22146 PubMedCrossRefGoogle Scholar
  47. Yu L, Zhang B, Szilvay GR, Sun R, Jänis J, Wang Z, Feng S, Xu H, Linder MB, Qiao M (2008) Protein HGFI from the edible mushroom Grifola frondosa is a novel 8 kDa class I hydrophobin that forms rodlets in compressed monolayers. Microbiology 154(6):1677–1685. doi: 10.1099/mic.0.2007/015263-0 PubMedCrossRefGoogle Scholar
  48. Zampieri F, Wösten HAB, Scholtmeijer K (2010) Creating surface properties using a palette of hydrophobins. Materials 3:4607–4625. doi: 10.3390/ma3094607 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Baolong Niu
    • 1
    • 2
  • Yanbo Gong
    • 3
  • Xianghua Gao
    • 1
    • 2
  • Haijin Xu
    • 4
  • Mingqiang Qiao
    • 4
  • Wenfeng Li
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Interface Science and Engineering in Advanced Materials, College of Materials Science and EngineeringTaiyuan University of Technology, Ministry of EducationTaiyuanPeople’s Republic of China
  2. 2.College of Materials Science and EngineeringTaiyuan University of TechnologyTaiyuanPeople’s Republic of China
  3. 3.College of Light Textile Engineering and ArtTaiyuan University of TechnologyTaiyuanPeople’s Republic of China
  4. 4.State Key Laboratory of Medicinal Chemical Biology, College of Life SciencesNankai UniversityTianjinPeople’s Republic of China

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