Biology:Hydrophobin

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Fungal hydrophobin
Hydrophobin.png
Structure of hydrophobin HFBII from Trichoderma reesei
Identifiers
SymbolHydrophobin_2
PfamPF06766
InterProIPR010636
PROSITEPDOC00739
SCOP21r2m / SCOPe / SUPFAM
OPM superfamily96
OPM protein1r2m
Hydrophobin
Identifiers
SymbolHydrophobin
PfamPF01185
InterProIPR001338

Hydrophobins are a group of small (~100 amino acids) cysteine-rich proteins that were discovered in filamentous fungi that are lichenized or not. Later similar proteins were also found in Bacteria.[1] Hydrophobins are known for their ability to form a hydrophobic (water-repellent) coating on the surface of an object.[2] They were first discovered and separated in Schizophyllum commune in 1991.[3] 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 hydrophilic:hydrophobic 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.[4]

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

Hydrophobins have been identified in lichens[5] as well as non-lichenized ascomycetes and basidiomycetes; whether they exist in other groups is not known.[6] 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.[7] Some family members contain multiple copies of the domain.

Hydrophobins have been found to be structurally and functionally similar to cerato-platanins, another group of small cysteine-rich proteins,[8] which also contain a high percentage of hydrophobic amino acids,[6] and are also associated with hyphal growth.[9][10]

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.[11][12]

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.[13] High proportion of acidic amino acids is thought to be an adaptation of proteins to high concentrations of salt.[14]

Structure

Hydrophobins are characterised by the presence of 8 conserved cysteine residues that form 4 disulphide bonds.[15] 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 amphipathic monolayers they form.[15][16] 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

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.[17] 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.[18] The formation of rodlets involves conformational changes[19] that lead to formation of an extremely robust β-sheet structure[20] that can only be depolymerised by treatment with strong acids.[21] The rodlets can spontaneously form ordered monolayers by lateral assembly, displaying a regular fibrillary morphology on hydrophobic:hydrophilic interfaces.[22] 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.[23]

Class II

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.[17] 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.[22] 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.[24]

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

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.[18] 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.[25]

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.[26] Instead, a region of the short Cys7-Cys8 loop, containing mainly uncharged polar residues, has been found to be critical for rodlet assembly.[15]

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.[27] 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.[23] 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

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.[16] 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.[28] 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.
  • Hydrophobins' 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,[29][30][31] drug delivery[32][33][34] and cell attachment[35][36][37] has been reported.

For more about the potential biotechnological applications of hydrophobins see Hektor & Scholtmeijer (2005)[38] and Cox & Hooley (2009).[39]

References

  1. ↑ "BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm". PNAS 110 (33): 13600–5. July 2013. doi:10.1073/pnas.1306390110. PMID 23904481. Bibcode: 2013PNAS..11013600H. 
  2. ↑ "Structural analysis of hydrophobins". Micron 39 (7): 773–84. October 2008. doi:10.1016/j.micron.2007.08.003. PMID 17875392. 
  3. ↑ "Hydrophobin Genes Involved in Formation of Aerial Hyphae and Fruit Bodies in Schizophyllum". The Plant Cell 3 (8): 793–799. August 1991. doi:10.1105/tpc.3.8.793. PMID 12324614. 
  4. ↑ "Solid-state NMR spectroscopy of functional amyloid from a fungal hydrophobin: a well-ordered β-sheet core amidst structural heterogeneity". Angewandte Chemie 51 (50): 12621–5. December 2012. doi:10.1002/anie.201205625. PMID 23125123. 
  5. ↑ Peter Döbbeler, Gerhard Rambold (2004). Contributions to Lichenology. Gebrüder Borntraeger Verlagsbuchhandlung. pp. 207. 
  6. ↑ 6.0 6.1 "Hydrophobins: multipurpose proteins". Annual Review of Microbiology 55: 625–46. 2001. doi:10.1146/annurev.micro.55.1.625. PMID 11544369. 
  7. ↑ "The hydrophobin HCf-1 of Cladosporium fulvum is required for efficient water-mediated dispersal of conidia". Fungal Genetics and Biology 32 (3): 159–68. April 2001. doi:10.1006/fgbi.2001.1263. PMID 11343402. 
  8. ↑ "Distribution and bioinformatic analysis of the cerato-platanin protein family in Dikarya". Mycologia 105 (6): 1479–88. 20 January 2017. doi:10.3852/13-115. PMID 23928425. 
  9. ↑ "The expression of the cerato-platanin gene is related to hyphal growth and chlamydospores formation in Ceratocystis platani". FEMS Microbiology Letters 327 (2): 155–63. February 2012. doi:10.1111/j.1574-6968.2011.02475.x. PMID 22136757. 
  10. ↑ "How a fungus escapes the water to grow into the air". Current Biology 9 (2): 85–8. January 1999. doi:10.1016/S0960-9822(99)80019-0. PMID 10021365. 
  11. ↑ "Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation". Genes & Development 5 (7): 1161–71. July 1991. doi:10.1101/gad.5.7.1161. PMID 2065971. 
  12. ↑ "Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora". Genes & Development 6 (12A): 2373–81. December 1992. doi:10.1101/gad.6.12a.2373. PMID 1459459. 
  13. ↑ "Genome and transcriptome sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations present and absent". BMC Genomics 14: 617. September 2013. doi:10.1186/1471-2164-14-617. PMID 24034603. 
  14. ↑ "Halophilic adaptation of enzymes". Extremophiles 4 (2): 91–8. April 2000. doi:10.1007/s007920050142. PMID 10805563. 
  15. ↑ 15.0 15.1 15.2 "Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS". Proceedings of the National Academy of Sciences of the United States of America 109 (14): E804–11. April 2012. doi:10.1073/pnas.1114052109. PMID 22308366. 
  16. ↑ 16.0 16.1 "Developmental regulation of fungal cell wall formation.". Annual Review of Phytopathology 32 (1): 413–37. September 1994. doi:10.1146/annurev.py.32.090194.002213. 
  17. ↑ 17.0 17.1 "Hydrophobins: proteins that change the nature of the fungal surface". Advances in Microbial Physiology Volume 38. 38. 1997. 1–45. doi:10.1016/S0065-2911(08)60154-X. ISBN 9780120277384. 
  18. ↑ 18.0 18.1 "Structural basis for rodlet assembly in fungal hydrophobins". Proceedings of the National Academy of Sciences of the United States of America 103 (10): 3621–6. March 2006. doi:10.1073/pnas.0505704103. PMID 16537446. Bibcode: 2006PNAS..103.3621K. 
  19. ↑ "A diversity of assembly mechanisms of a generic amyloid fold". Molecular Cell 43 (1): 8–18. July 2011. doi:10.1016/j.molcel.2011.05.012. PMID 21726806. 
  20. ↑ "Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia". Journal of Bacteriology 140 (3): 1063–70. December 1979. doi:10.1128/jb.140.3.1063-1070.1979. PMID 160407. 
  21. ↑ "Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi.". Archives of Microbiology 159 (4): 330–5. April 1993. doi:10.1007/BF00290915. Bibcode: 1993ArMic.159..330D. 
  22. ↑ 22.0 22.1 "Two forms and two faces, multiple states and multiple uses: Properties and applications of the self-assembling fungal hydrophobins". Biopolymers 100 (6): 601–12. November 2013. doi:10.1002/bip.22259. PMID 23913717. 
  23. ↑ 23.0 23.1 "Analysis of the structure and conformational states of DewA gives insight into the assembly of the fungal hydrophobins". Journal of Molecular Biology 425 (2): 244–56. January 2013. doi:10.1016/j.jmb.2012.10.021. PMID 23137797. 
  24. ↑ "Self-assembled hydrophobin protein films at the air-water interface: structural analysis and molecular engineering". Biochemistry 46 (9): 2345–54. March 2007. doi:10.1021/bi602358h. PMID 17297923. 
  25. ↑ "Common core structure of amyloid fibrils by synchrotron X-ray diffraction". Journal of Molecular Biology 273 (3): 729–39. October 1997. doi:10.1006/jmbi.1997.1348. PMID 9356260. 
  26. ↑ "The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity". Journal of Molecular Biology 382 (3): 708–20. October 2008. doi:10.1016/j.jmb.2008.07.034. PMID 18674544. 
  27. ↑ "Backbone and sidechain ¹H, ¹³C and ¹⁵N chemical shift assignments of the hydrophobin DewA from Aspergillus nidulans". Biomolecular NMR Assignments 6 (1): 83–6. April 2012. doi:10.1007/s12104-011-9330-5. PMID 21845363. 
  28. ↑ "Surface functionalization of carbon nanomaterials by self-assembling hydrophobin proteins". Biopolymers 99 (1): 84–94. January 2013. doi:10.1002/bip.22146. PMID 23097233. 
  29. ↑ "Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems". Biochemistry 43 (37): 11873–82. September 2004. doi:10.1021/bi0488202. PMID 15362873. 
  30. ↑ "Extraction of endoglucanase I (Ce17B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system". Journal of Chromatography A 943 (1): 55–62. January 2002. doi:10.1016/S0021-9673(01)01433-9. PMID 11820281. 
  31. ↑ "Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana". Plant Physiology 152 (2): 622–33. February 2010. doi:10.1104/pp.109.149021. PMID 20018596. 
  32. ↑ "Use of hydrophobins in formulation of water insoluble drugs for oral administration". Colloids and Surfaces. B, Biointerfaces 75 (2): 526–31. February 2010. doi:10.1016/j.colsurfb.2009.09.030. PMID 19836932. 
  33. ↑ "Cellular interactions of surface modified nanoporous silicon particles". Nanoscale 4 (10): 3184–92. May 2012. doi:10.1039/c2nr30397c. PMID 22508528. Bibcode: 2012Nanos...4.3184B. 
  34. ↑ "Intravenous delivery of hydrophobin-functionalized porous silicon nanoparticles: stability, plasma protein adsorption and biodistribution". Molecular Pharmaceutics 9 (3): 654–63. March 2012. doi:10.1021/mp200611d. PMID 22277076. 
  35. ↑ "Expression of a fungal hydrophobin in the Saccharomyces cerevisiae cell wall: effect on cell surface properties and immobilization". Applied and Environmental Microbiology 68 (7): 3385–91. July 2002. doi:10.1128/AEM.68.7.3385-3391.2002. PMID 12089019. Bibcode: 2002ApEnM..68.3385N. 
  36. ↑ "Expression and characterization of hydrophobin HGFI fused with the cell-specific peptide TPS in Pichia pastoris". Protein Expression and Purification 83 (1): 92–7. May 2012. doi:10.1016/j.pep.2012.03.004. PMID 22440542. 
  37. ↑ "Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells". Acta Biomaterialia 8 (3): 1037–47. March 2012. doi:10.1016/j.actbio.2011.11.022. PMID 22154865. 
  38. ↑ "Hydrophobins: proteins with potential". Current Opinion in Biotechnology 16 (4): 434–9. August 2005. doi:10.1016/j.copbio.2005.05.004. PMID 15950452. 
  39. ↑ "Hydrophobins: new prospects for biotechnology.". Fungal Biology Reviews 23 (1–2): 40–7. February 2009. doi:10.1016/j.fbr.2009.09.001. 

Further reading

  • Scholtmeijer K (2000). Expression and engineering of hydrophobin genes (Ph.D. thesis). University of Groningen.
  • "Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile". The Journal of Biological Chemistry 279 (1): 534–9. January 2004. doi:10.1074/jbc.M309650200. PMID 14555650. 
  • "Hydrophobins, the fungal coat unravelled". Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes 1469 (2): 79–86. September 2000. doi:10.1016/S0304-4157(00)00002-2. PMID 10998570. 
  • "Surface hydrophobin prevents immune recognition of airborne fungal spores". Nature 460 (7259): 1117–21. August 2009. doi:10.1038/nature08264. PMID 19713928. Bibcode: 2009Natur.460.1117A. 


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



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