Hydrophobin

Hydrophobins are characterised by the presence of 8 conserved cysteine residues that form 4 disulphide bonds.{{cite journal | vauthors = Macindoe I, Kwan AH, Ren Q, Morris VK, Yang W, Mackay JP, Sunde M | title = Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 14 | pages = E804–11 | date = April 2012 | pmid = 22308366 | pmc = 3325668 | doi = 10.1073/pnas.1114052109 | doi-access = free }} 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.{{cite journal | vauthors = Wessels JG | title = Developmental regulation of fungal cell wall formation. | journal = Annual Review of Phytopathology | date = September 1994 | volume = 32 | issue = 1 | pages = 413–37 | doi = 10.1146/annurev.py.32.090194.002213 | bibcode = 1994AnRvP..32..413W }} 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 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.{{cite book | vauthors = Wessels JG | title = Advances in Microbial Physiology Volume 38 | chapter = Hydrophobins: proteins that change the nature of the fungal surface | journal = Advances in Microbial Physiology | volume = 38 | pages = 1–45 | date = 1997 | pmid = 8922117 | doi = 10.1016/S0065-2911(08)60154-X | isbn = 9780120277384 }} 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.{{cite journal | vauthors = Kwan AH, Winefield RD, Sunde M, Matthews JM, Haverkamp RG, Templeton MD, Mackay JP | title = Structural basis for rodlet assembly in fungal hydrophobins | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 103 | issue = 10 | pages = 3621–6 | date = March 2006 | pmid = 16537446 | pmc = 1533775 | doi = 10.1073/pnas.0505704103 | bibcode = 2006PNAS..103.3621K | doi-access = free }} The formation of rodlets involves conformational changes{{cite journal | vauthors = Eichner T, Radford SE | title = A diversity of assembly mechanisms of a generic amyloid fold | journal = Molecular Cell | volume = 43 | issue = 1 | pages = 8–18 | date = July 2011 | pmid = 21726806 | doi = 10.1016/j.molcel.2011.05.012 | doi-access = free }} that lead to formation of an extremely robust β-sheet structure{{cite journal | vauthors = Beever RE, Redgwell RJ, Dempsey GP | title = Purification and chemical characterization of the rodlet layer of Neurospora crassa conidia | journal = Journal of Bacteriology | volume = 140 | issue = 3 | pages = 1063–70 | date = December 1979 | pmid = 160407 | pmc = 216753 | url = | doi = 10.1128/jb.140.3.1063-1070.1979 }} that can only be depolymerised by treatment with strong acids.{{cite journal | vauthors = de Vries OM, Fekkes MP, Wösten HA, Wessels JG | title = Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi. | journal = Archives of Microbiology | date = April 1993 | volume = 159 | issue = 4 | pages = 330–5 | doi = 10.1007/BF00290915 | bibcode = 1993ArMic.159..330D | s2cid = 25891213 }} The rodlets can spontaneously form ordered monolayers by lateral assembly, displaying a regular fibrillary morphology on hydrophobic:hydrophilic interfaces.{{cite journal | vauthors = Ren Q, Kwan AH, Sunde M | title = Two forms and two faces, multiple states and multiple uses: Properties and applications of the self-assembling fungal hydrophobins | journal = Biopolymers | volume = 100 | issue = 6 | pages = 601–12 | date = November 2013 | pmid = 23913717 | doi = 10.1002/bip.22259 }} 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.{{cite journal | vauthors = Morris VK, Kwan AH, Sunde M | title = Analysis of the structure and conformational states of DewA gives insight into the assembly of the fungal hydrophobins | journal = Journal of Molecular Biology | volume = 425 | issue = 2 | pages = 244–56 | date = January 2013 | pmid = 23137797 | doi = 10.1016/j.jmb.2012.10.021 }}

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. 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. 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.{{cite journal | vauthors = Szilvay GR, Paananen A, Laurikainen K, Vuorimaa E, Lemmetyinen H, Peltonen J, Linder MB | title = Self-assembled hydrophobin protein films at the air-water interface: structural analysis and molecular engineering | journal = Biochemistry | volume = 46 | issue = 9 | pages = 2345–54 | date = March 2007 | pmid = 17297923 | doi = 10.1021/bi602358h }}

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

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. 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.{{cite journal | vauthors = Sunde M, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CC | title = Common core structure of amyloid fibrils by synchrotron X-ray diffraction | journal = Journal of Molecular Biology | volume = 273 | issue = 3 | pages = 729–39 | date = October 1997 | pmid = 9356260 | doi = 10.1006/jmbi.1997.1348 }}

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.{{cite journal | vauthors = Kwan AH, Macindoe I, Vukasin PV, Morris VK, Kass I, Gupte R, Mark AE, Templeton MD, Mackay JP, Sunde M | title = The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity | journal = Journal of Molecular Biology | volume = 382 | issue = 3 | pages = 708–20 | date = October 2008 | pmid = 18674544 | doi = 10.1016/j.jmb.2008.07.034 }} Instead, a region of the short Cys7-Cys8 loop, containing mainly uncharged polar residues, has been found to be critical for rodlet assembly.

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.{{cite journal | vauthors = Morris VK, Kwan AH, Mackay JP, Sunde M | title = Backbone and sidechain ¹H, ¹³C and ¹⁵N chemical shift assignments of the hydrophobin DewA from Aspergillus nidulans | journal = Biomolecular NMR Assignments | volume = 6 | issue = 1 | pages = 83–6 | date = April 2012 | pmid = 21845363 | doi = 10.1007/s12104-011-9330-5 | s2cid = 29402126 }} 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. 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.

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. 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.{{cite journal | vauthors = Yang W, Ren Q, Wu YN, Morris VK, Rey AA, Braet F, Kwan AH, Sunde M | title = Surface functionalization of carbon nanomaterials by self-assembling hydrophobin proteins | journal = Biopolymers | volume = 99 | issue = 1 | pages = 84–94 | date = January 2013 | pmid = 23097233 | doi = 10.1002/bip.22146 }} 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,{{cite journal | vauthors = Linder MB, Qiao M, Laumen F, Selber K, Hyytiä T, Nakari-Setälä T, Penttilä ME | title = Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems | journal = Biochemistry | volume = 43 | issue = 37 | pages = 11873–82 | date = September 2004 | pmid = 15362873 | doi = 10.1021/bi0488202 }}{{cite journal | vauthors = Collén A, Penttilä M, Stålbrand H, Tjerneld F, Veide A | title = Extraction of endoglucanase I (Ce17B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system | journal = Journal of Chromatography A | volume = 943 | issue = 1 | pages = 55–62 | date = January 2002 | pmid = 11820281 | doi = 10.1016/S0021-9673(01)01433-9 }}{{cite journal | vauthors = Joensuu JJ, Conley AJ, Lienemann M, Brandle JE, Linder MB, Menassa R | title = Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana | journal = Plant Physiology | volume = 152 | issue = 2 | pages = 622–33 | date = February 2010 | pmid = 20018596 | pmc = 2815860 | doi = 10.1104/pp.109.149021 }} drug delivery{{cite journal | vauthors = Haas Jimoh Akanbi M, Post E, Meter-Arkema A, Rink R, Robillard GT, Wang X, Wösten HA, Scholtmeijer K | title = Use of hydrophobins in formulation of water insoluble drugs for oral administration | journal = Colloids and Surfaces B: Biointerfaces | volume = 75 | issue = 2 | pages = 526–31 | date = February 2010 | pmid = 19836932 | doi = 10.1016/j.colsurfb.2009.09.030 }}{{cite journal | vauthors = Bimbo LM, Sarparanta M, Mäkilä E, Laaksonen T, Laaksonen P, Salonen J, Linder MB, Hirvonen J, Airaksinen AJ, Santos HA | title = Cellular interactions of surface modified nanoporous silicon particles | journal = Nanoscale | volume = 4 | issue = 10 | pages = 3184–92 | date = May 2012 | pmid = 22508528 | doi = 10.1039/c2nr30397c | bibcode = 2012Nanos...4.3184B }}{{cite journal | vauthors = Sarparanta M, Bimbo LM, Rytkönen J, Mäkilä E, Laaksonen TJ, Laaksonen P, Nyman M, Salonen J, Linder MB, Hirvonen J, Santos HA, Airaksinen AJ | title = Intravenous delivery of hydrophobin-functionalized porous silicon nanoparticles: stability, plasma protein adsorption and biodistribution | journal = Molecular Pharmaceutics | volume = 9 | issue = 3 | pages = 654–63 | date = March 2012 | pmid = 22277076 | doi = 10.1021/mp200611d }} and cell attachment{{cite journal | vauthors = Nakari-Setälä T, Azeredo J, Henriques M, Oliveira R, Teixeira J, Linder M, Penttilä M | title = Expression of a fungal hydrophobin in the Saccharomyces cerevisiae cell wall: effect on cell surface properties and immobilization | journal = Applied and Environmental Microbiology | volume = 68 | issue = 7 | pages = 3385–91 | date = July 2002 | pmid = 12089019 | pmc = 126783 | doi = 10.1128/AEM.68.7.3385-3391.2002 | bibcode = 2002ApEnM..68.3385N }}{{cite journal | vauthors = Niu B, Huang Y, Zhang S, Wang D, Xu H, Kong D, Qiao M | title = Expression and characterization of hydrophobin HGFI fused with the cell-specific peptide TPS in Pichia pastoris | journal = Protein Expression and Purification | volume = 83 | issue = 1 | pages = 92–7 | date = May 2012 | pmid = 22440542 | doi = 10.1016/j.pep.2012.03.004 }}{{cite journal | vauthors = Boeuf S, Throm T, Gutt B, Strunk T, Hoffmann M, Seebach E, Mühlberg L, Brocher J, Gotterbarm T, Wenzel W, Fischer R, Richter W | title = Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells | journal = Acta Biomaterialia | volume = 8 | issue = 3 | pages = 1037–47 | date = March 2012 | pmid = 22154865 | doi = 10.1016/j.actbio.2011.11.022 | url = https://publikationen.bibliothek.kit.edu/1000040555/150160204 }} has been reported.

For more about the potential biotechnological applications of hydrophobins see Hektor & Scholtmeijer (2005){{cite journal | vauthors = Hektor HJ, Scholtmeijer K | title = Hydrophobins: proteins with potential | journal = Current Opinion in Biotechnology | volume = 16 | issue = 4 | pages = 434–9 | date = August 2005 | pmid = 15950452 | doi = 10.1016/j.copbio.2005.05.004 }} and Cox & Hooley (2009).{{cite journal | vauthors = Cox PW, Hooley P | title = Hydrophobins: new prospects for biotechnology. | journal = Fungal Biology Reviews | date = February 2009 | volume = 23 | issue = 1–2 | pages = 40–7 | doi = 10.1016/j.fbr.2009.09.001 | bibcode = 2009FunBR..23...40C | hdl = 2436/117149 | hdl-access = free }}

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• {{Cite thesis | degree= Ph.D. | title = Expression and engineering of hydrophobin genes | url = http://irs.ub.rug.nl/ppn/240088549 | author = Scholtmeijer K | year = 2000 | publisher = University of Groningen }}
• {{cite journal | vauthors = Hakanpää J, Paananen A, Askolin S, Nakari-Setälä T, Parkkinen T, Penttilä M, Linder MB, Rouvinen J | title = Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile | journal = The Journal of Biological Chemistry | volume = 279 | issue = 1 | pages = 534–9 | date = January 2004 | pmid = 14555650 | doi = 10.1074/jbc.M309650200 | doi-access = free }}
• {{cite journal | vauthors = Wösten HA, de Vocht ML | title = Hydrophobins, the fungal coat unravelled | journal = Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes | volume = 1469 | issue = 2 | pages = 79–86 | date = September 2000 | pmid = 10998570 | doi = 10.1016/S0304-4157(00)00002-2 }}
• {{cite journal | vauthors = Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, Elluru SR, Clavaud C, Paris S, Brakhage AA, Kaveri SV, Romani L, Latgé JP | title = Surface hydrophobin prevents immune recognition of airborne fungal spores | journal = Nature | volume = 460 | issue = 7259 | pages = 1117–21 | date = August 2009 | pmid = 19713928 | doi = 10.1038/nature08264 | bibcode = 2009Natur.460.1117A | s2cid = 4333767 }}

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Category:Protein families

Category:Proteins