Hydrophobin

From Wikipedia, the free encyclopedia
Jump to: navigation, search
Fungal hydrophobin
Hydrophobin.png
Structure of hydrophobin HFBI from Trichoderma reesei
Identifiers
Symbol Hydrophobin_2
Pfam PF06766
InterPro IPR010636
PROSITE PDOC00739
SCOP 1r2m
SUPERFAMILY 1r2m
OPM superfamily 104
OPM protein 1r2m
Hydrophobin
Identifiers
Symbol Hydrophobin
Pfam PF01185
InterPro IPR001338

Hydrophobins are a group of small (~100 amino acids) cysteine-rich proteins that are expressed only by filamentous fungi. They are known for their ability to form a hydrophobic (water-repellent) coating on the surface of an object.[1] They were first discovered and separated in Schizophyllum commune in 1991.[2] Based on differences in hydropathy patterns and biophysical properties, they can be divided into two categories: class I and class II. Hydrophobins can self-assemble into a monolayer on hydrophobic:hydrophilic interfaces such as a water:air interface. Class I monolayer contains the same core structure as amyloid fibrils, and is positive to Congo red and thioflavin T. The monolayer formed by class I hydrophobins has a highly ordered structure, and can only be dissociated by concentrated trifluoroacetate or formic acid. Monolayer assembly involves large structural rearrangements with respect to the monomer.[3]

Fungi make complex aerial structures and spores even in aqueous environments.

Hydrophobins have been identified in ascomycetes and basidiomycetes; whether they exist in other groups is not known.[4] Hydrophobins are generally found on the outer surface of conidia and of the hyphal wall, and may be involved in mediating contact and communication between the fungus and its environment.[5] Some family members contain multiple copies of the domain.

This family of proteins includes the rodlet proteins of Neurospora crassa (gene eas) and Emericella nidulans (gene rodA), these proteins are the main component of the hydrophobic sheath covering the surface of many fungal spores.[6][7]

Genomic sequencing of two fungi from dry or salty environments (Wallemia sebi and W. ichthyophaga) revealed that these species contain predicted hydrophobins with unusually high proportion of acidic amino acids and therefore with potentially novel characteristics.[8] High proportion of acidic amino acids is thought to be an adaptation of proteins to high concentrations of salt.[9]

Hydrophobin structure[edit]

Hydrophobins are characterised by the presence of 8 conserved cysteine residues that form 4 disulphide bonds.[10] They are able to reverse the wettability of surfaces by spontaneous self-assembly of the monomeric proteins into amphipathic monolayers at hydrophobic:hydrophilic surfaces. Despite this common feature, hydrophobins are subdivided into two classes based on differences on their monomeric structure, such as the spacing between the cysteine residues, and based on the different physicochemical properties of the amphipatic monolayers they form [10][11] Extensive structural analyses of individual hydrophobins from the two classes have elucidated that the morphological and physical differences between the class I and class II polymer forms are the results of significant structural differences at the monomer-assembly level.

Class I[edit]

Class I hydrophobins are characterised by having a quite diverse amino acid sequence between different types (with exception of the conserved cysteine residues), and compared to class II, they have long, varied inter-cysteine spacing.[12] They form rodlets which have been identified as functional amyloids due to their amyloid-like characteristics as seen in X-ray diffraction studies and confirmed by their capacity to bind to amyloid-specific dyes such as Congo red and Thioflavin T.[13] The formation of rodlets involves conformational changes [14] that lead to formation of an extremely robust β-sheet structure [15] that can only be depolymerised by treatment with strong acids.[16] The rodlets can spontaneously form ordered monolayers by lateral assembly, displaying a regular fibrillary morphology on hydrophobic:hydrophilic interfaces.[17] The most well characterised class I hydrophobin is EAS, which coats the spores of the fungus Neurospora crassa, followed by characterisation of DewA from Aspergillus nidulans.[18]

Class II[edit]

Class II hydrophobins have overall a more conserved amino acid sequence between the different types and, contrary to class, I they have short, regular inter-cysteine spacing.[12] Opposite to class I, the class II hydrophobins monolayer formed at hydrophobic:hydrophilic interfaces is not fibrillar and it is not associated with formation of amyloid-structures, nor with large conformational changes.[17] Nonetheless, high resolution atomic-force microscopy studies revealed the formation of a notable hexagonal repeating pattern over surfaces coated with the class II hydrophobin HBFI, meaning that these proteins are also able to form an ordered network in surface films.[19]

The crystal structures or HFBI and HFBII from Trichoderma reesei were the first class II hydrophobins to be determined.

Rodlet self-assembly of class I hydrophobins[edit]

There is special interest in understanding the mechanism underlying class I monomers self-assembly that leads to formation of tough, ordered amphipathic rodlet monolayers, due to their intrinsic properties and due to substantial information available from several characterisation studies of the class I hydrophobins EAS and DewA. These mechanisms have been greatly studied by targeted mutagenesis in an effort to identify the key amino acid sequence regions driving rodlet self-assembly. A model for the monomeric form of EAS was proposed by Kwan et al. (2006) from structural data obtained from NMR spectroscopy and X-ray diffraction experiments that indicated the presence of four-stranded, antiparallel β-barrel core structure in EAS that allows monomer linking through backbone H-bonding.[13] There are secondary elements around this β-barrel core like the Cys3-Cys4 and Cys7-Cys8 loops. This model is consistent with the amyloid-like structure that class I rodlets form, in which the β-strands are oriented perpendicular to the cross-β scaffold axis of the fibre.[20]

Site-directed mutagenesis of EAS has given insights into the specific structural changes responsible for self-assembly of monomers into rodlets and subsequent formation of amphipathic monolayer in hydrophobic:hydrophilic interfaces. Kwan et al. (2008) reported that the long hydrophobic Cys3-Cys4 loop is not required for rodlet assembly because its deletion does not affect the folding and physical properties of the monomeric protein, neither the morphology of the polymeric rodlet form.[21] Instead, a region of the short Cys7-Cys8 loop, containing mainly uncharged polar residues, has been found to be critical for rodlet assembly.[10]

Characterization of EAS secondary elements involved in rodlet assembly have given insights into the mechanism behind class I hydrophobins self-assembly, but important structural differences with DewA, another class I hydrophobin, suggest that the mechanisms driving rodlet assembly vary among different types of hydrophobins. Like EAS, DewA also has a β-barrel core structure, but it differs significantly from it because of its considerable content of helical secondary elements.[22] A unique feature of DewA is its capacity to exist as two types of conformers in solution, both able to form rodlet assemblies but at different rates.[18] Despite these differences in structural and self-assembly mechanisms, both EAS and DewA form robust fibrillar monolayers, meaning that there must exist several pathways, protein sequences and tertiary conformations able to self-assemble into amphipathic monolayers. Further characterisation of both EAS and DewA and their rodlet self-assembly mechanisms will open up opportunities for rational design of hydrophobins with novel biotechnological applications.

Potentiality for use[edit]

Since the very first studies that gave insights into the properties of hydrophobins, these small proteins have been regarded as great candidates for technological use.[23] The detailed understanding of the molecular mechanisms underlying hydrophobin self-assembly into amphipathic monolayer in hydrophobic:hydrophilic interfaces is of great academic interest but mainly of commercial interest. This is because a deep understanding of the elements driving these mechanisms would allow engineering of hydrophobins (or other biomolecules) for nano and biotechnological applications. An example is that the hydrophobin-coating of carbon nanotubes was found to increase their solubility and reduce their toxicity, a finding that increases the prospects of carbon nanotubes to be used as vehicles for drug delivery.[24] Other areas of potential use of hydrophobins include:

  • Fabrication and coating of nanodevices and medical implants to increase biocompatibility.
  • Emulsifiers in food industry and personal care products.
  • Class I high stability can be very useful in the coating of surfaces of prolonged use or under harsh conditions.
  • The easy dissociation of a class II hydrophobin monolayer might be desirable and this can easily be achieved by the use of detergents and alcohols.
  • The use of hydrophobins in protein purification,[25][26][27] drug delivery [28][29][30] and cell attachment[31][32][33] has been reported.

For more about the potential biotechnological applications of hydrophobins see Hektor & Scholtmeijer (2005)[34] and Cox & Hooley (2009)[35]

References[edit]

  1. ^ Sunde M, Kwan AH, Templeton MD, Beever RE, Mackay JP (October 2008). "Structural analysis of hydrophobins". Micron. 39 (7): 773–84. PMID 17875392. doi:10.1016/j.micron.2007.08.003. 
  2. ^ 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. 3 (8): 793–799. PMC 160046Freely accessible. PMID 12324614. doi:10.1105/tpc.3.8.793. 
  3. ^ Morris V. K.; Linser R.; Wilde K. L.; Duff A. P.; Sunde M.; Kwan A. H. (2012). "Solid-State NMR Spectroscopy of Functional Amyloid from a Fungal Hydrophobin: A Well-Ordered β-Sheet Core Amidst Structural Heterogeneity". Angew. Chem. Int. Ed. 51: 12621–12625. doi:10.1002/anie.201205625. 
  4. ^ Wösten (2001). "Hydrophobins: multipurpose proteins". Annual Review of Microbiology. 55: 625–646. PMID 11544369. doi:10.1146/annurev.micro.55.1.625. 
  5. ^ Whiteford JR, Spanu PD (2001). "The hydrophobin HCf-1 of Cladosporium fulvum is required for efficient water-mediated dispersal of conidia". Fungal Genet. Biol. 32 (3): 159–168. PMID 11343402. doi:10.1006/fgbi.2001.1263. 
  6. ^ Stringer MA, Dean RA, Sewall TC, Timberlake WE (July 1991). "Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation". Genes Dev. 5 (7): 1161–71. PMID 2065971. doi:10.1101/gad.5.7.1161. 
  7. ^ Lauter FR, Russo VE, Yanofsky C (December 1992). "Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora". Genes Dev. 6 (12A): 2373–81. PMID 1459459. doi:10.1101/gad.6.12a.2373. 
  8. ^ Zajc, J.; Liu, Y.; Dai, W.; Yang, Z.; Hu, J.; Gostin Ar, C.; Gunde-Cimerman, N. (2013). "Genome and transcriptome sequencing of the halophilic fungus Wallemia ichthyophaga: Haloadaptations present and absent". BMC Genomics. 14: 617. PMC 3849046Freely accessible. PMID 24034603. doi:10.1186/1471-2164-14-617. 
  9. ^ Madern, D.; Ebel, C.; Zaccai, G. (2000). "Halophilic adaptation of enzymes". Extremophiles : life under extreme conditions. 4 (2): 91–98. PMID 10805563. doi:10.1007/s007920050142. 
  10. ^ a b c Macindoe, I. et al., 2012. Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS. Proceedings of the National Academy of Sciences, 109(14), pp. E804-E811
  11. ^ Wessels, J., 1994. Developmental Regulation of Fungal Cell Wall Formation. Annual Review Phytopathology, 32(1), pp. 413-437
  12. ^ a b Wessels, J., 1996. Hydrophobins: proteins that change the nature of the fungal surface. Advances in microbial physiology, Volume 38, pp. 1-45
  13. ^ a b Kwan, A. et al., 2006. Structural basis for rodlet assembly in fungal hydrophobins. Proceedings of the National Academy of Sciences, 103(10), pp. 3621-3626
  14. ^ Eichner, T. & Radford, S. E., 2011. A diversity of assembly mechanisms of a generic amyloid fold. Molecular Cell, Volume 43, pp. 8-18
  15. ^ Wösten, H. & Wessels, J., 1979. Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia. Journal of Bacteriology, Volume 140, pp. 1063-1070
  16. ^ de Vries, O. M., Fekkes, M. P., Wösten, H. A. & Wessels, J. G., 1993. Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi. Archives of Microbiology, 159(4), pp. 330-335
  17. ^ a b Ren, Q., Kwan, A. & Sunde, M., 2013. Two forms and two faces, multiple states and multiple uses: Properties and applications of the self-assembling fungal hydrophobins. Biopolymers, 100(6), pp. 601-612
  18. ^ a b Morris, V., Kwan, A. & Sunde, M., 2012. Analysis of the Structure and Conformational States of DewA Gives Insight into the Assembly of the Fungal Hydrophobins. Journal of Molecular Biology, Volume 452, pp. 245-256
  19. ^ Szilvay, G. et al., 2007. Self-Assembled Hydrophobin Protein Films at the Air−Water Interface: Structural Analysis and Molecular Engineering. Biochemistry, 46(9), pp. 2345-2354
  20. ^ Sunde, M. et al., 1997. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. Journal of Molecular Biology, 273(3), pp. 729-739
  21. ^ Kwan, A. et al., 2008. The Cys3–Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity. Journal of Molecular Biology, 382(3), pp. 708-720
  22. ^ Morris, V., Kwan, A., Mackay, J. & Sunde, M., 2011. Backbone and sidechain 1H, 13C and 15N chemical shift assignments of the hydrophobin DewA from Aspergillus nidulans. Biomolecular NMR assignments, 6(1), pp. 83-86
  23. ^ Wessels, J., 1994. Developmental Regulation of Fungal Cell Wall Formation.. Annual Review Phytopathology, 32(1), pp. 413-437
  24. ^ Yang, W. et al., 2012. Surface functionalization of carbon nanomaterials by self-assembling hydrophobin proteins. Biopolymers, 99(1), pp. 84-94
  25. ^ Lnder, M. et al., 2004. Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems. Biochemistry, 43(37), pp. 11873-11882
  26. ^ Collén, A. et al., 2002. Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)–phosphate aqueous two-phase system. Journal of Chromatography A, 943(1), pp. 55-62
  27. ^ Joensuu, J. et al., 2009. Hydrophobin Fusions for High-Level Transient Protein Expression and Purification in Nicotiana benthamiana. Plant Physiology, 152(2), pp. 622-633
  28. ^ Akanbi, M. et al., 2010. Use of hydrophobins in formulation of water insoluble drugs for oral administration. Colloids and Surfaces B: Biointerfaces, 75(2), pp. 526-531
  29. ^ Bimbo, L. et al., 2012. Cellular interactions of surface modified nanoporous silicon particles. Nanoscale, 4(10), pp. 3184-3192
  30. ^ Sarparanta, M. et al., 2012. Intravenous Delivery of Hydrophobin-Functionalized Porous Silicon Nanoparticles: Stability, Plasma Protein Adsorption and Biodistribution. Mol. Pharmaceutics, 9(3), pp. 654-663
  31. ^ Nakari-Setala, T. et al., 2002. Expression of a Fungal Hydrophobin in the Saccharomyces cerevisiae Cell Wall: Effect on Cell Surface Properties and Immobilization. Applied and Environmental Microbiology, 68(7), pp. 3385-3391
  32. ^ Niu, B. et al., 2012. Expression and characterization of hydrophobin HGFI fused with the cell-specific peptide TPS in Pichia pastoris. Protein Expression and Purification, 83(1), pp. 92-97
  33. ^ Boeuf, S. et al., 2012. Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells. Acta Biomaterialia, 8(3), pp. 1037-1047
  34. ^ Hektor, H. & Scholtmeijer, K., 2005. Hydrophobins: proteins with potential. Current Opinion in Biotechnology, 16(4), pp. 434-439
  35. ^ Cox, P. & Hooley, P., 2009. Hydrophobins: New prospects for biotechnology. Fungal Biology Reviews, 23(1), pp. 40-47

Further reading[edit]

  • Scholtmeijer K (2000). Expression and engineering of hydrophobin genes (Ph.D. thesis). University of Groningen. 
  • Hakanpää J, Paananen A, Askolin S, Nakari-Setälä T, Parkkinen T, Penttilä M, Linder MB, Rouvinen J (January 2004). "Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile". J. Biol. Chem. 279 (1): 534–9. PMID 14555650. doi:10.1074/jbc.M309650200. 
  • Wösten HA, de Vocht ML (September 2000). "Hydrophobins, the fungal coat unravelled". Biochim. Biophys. Acta. 1469 (2): 79–86. PMID 10998570. doi:10.1016/S0304-4157(00)00002-2. 
  • Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, Elluru SR, Clavaud C, Paris S, Brakhage AA, Kaveri SV, Romani L, Latgé JP (August 2009). "Surface hydrophobin prevents immune recognition of airborne fungal spores". Nature. 460 (7259): 1117–21. PMID 19713928. doi:10.1038/nature08264. 

This article incorporates text from the public domain Pfam and InterPro IPR001338