The impact of recombinant fusion-hydrophobin coated surfaces on E. coli and natural mixed culture biofilm formation

The impact of increased surface hydrophobicity on biofilms regarding retardation, repulsion, or attraction was studied with hydrophobin modified glass substrata. Recombinantly produced fungal hydrophobins forming self-assembled monolayers were used as the surface coating. The adsorption dynamics of hydrophobins were analysed with a quartz crystal microbalance which showed the surface coating to be rapid and stable. The change of surface wettability was determined by water contact angle measurements and demonstrated an increase in hydrophobicity in range of 60-62°. The homogeneity of the monolayers was demonstrated by immunofluorescence microscopy. Atomic force microscopy was applied to visualise the uniform texture of the coated materials. The hydrophobin coatings had no impact on different biofilms in terms of spatial distribution, cell numbers, and population composition. In consequence, hydrophobicity might not represent an important parameter for biofilm formation. Nevertheless, recombinant hydrophobins are suitable for large scale surface modification and functionalization with bioactive molecules.     

Surface functionalization of carbon nanomaterials by self‐assembling hydrophobin proteins

Class I fungal hydrophobins are small surface‐active proteins that self‐assemble to form amphipathic monolayers composed of amyloid‐like rodlets. The monolayers are extremely robust and can adsorb onto both hydrophobic and hydrophilic surfaces to reverse their wettability. This adherence is particularly strong for hydrophobic materials. In this report, we show that the class I hydrophobins EAS and HYD3 can self‐assemble to form a single‐molecule thick coating on a range of nanomaterials, including single‐walled carbon nanotubes (SWCNTs), graphene sheets, highly oriented pyrolytic graphite, and mica. Moreover, coating by class I hydrophobin results in a stable, dispersed preparation of SWCNTs in aqueous solutions. No cytotoxicity is detected when hydrophobin or hydrophobin‐coated SWCNTs are incubated with Caco‐2 cells in vitro. In addition, we are able to specifically introduce covalently linked chemical moieties to the hydrophilic side of the rodlet monolayer. Hence, class I hydrophobins provide a simple and effective strategy for controlling the surfaces of a range of materials at a molecular level and exhibit strong potential for biomedical applications. © 2012 Wiley Periodicals, Inc.     

Analysis of the Structure and Conformational States of DewA Gives Insight into the Assembly of the Fungal Hydrophobins

The hydrophobin DewA from the fungus Aspergillus nidulans is a highly surface-active protein that spontaneously self-assembles into amphipathic monolayers at hydrophobic:hydrophilic interfaces. These monolayers are composed of fibrils that are a form of functional amyloid. While there has been significant interest in the use of DewA for a variety of surface coatings and as an emulsifier in biotechnological applications, little is understood about the structure of the protein or the mechanism of self-assembly. We have solved the solution NMR structure of DewA. While the pattern of four disulfide bonds that is a defining feature of hydrophobins is conserved, the arrangement and composition of secondary-structure elements in DewA are quite different to what has been observed in other hydrophobin structures. In addition, we demonstrate that DewA populates two conformations in solution, both of which are assembly competent. One conformer forms a dimer at high concentrations, but this dimer is off-pathway to fibril formation and may represent an assembly control mechanism. These data highlight the structural differences between fibril-forming hydrophobins and those that form amorphous monolayers. This work will open up new opportunities for the engineering of hydrophobins with novel biotechnological applications.     

Solution structure and interface‐driven self‐assembly of NC2, a new member of the Class II hydrophobin proteins

Hydrophobins are fungal proteins that self‐assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin‐based applications. Proteins 2014; 82:990–1003. © 2013 Wiley Periodicals, Inc.     

Crystal structures of hydrophobin HFBII in the presence of detergent implicate the formation of fibrils and monolayer films

Hydrophobins are small, amphiphilic proteins secreted by filamentous fungi. Their functionality arises from a patch of hydrophobic residues on the protein surface. Spontaneous self-assembly of hydrophobins leads to the formation of an amphiphilic layer that remarkably reduces the surface tension of water. We have determined by x-ray diffraction two new crystal structures of Trichoderma reesei hydrophobin HFBII in the presence of a detergent. The monoclinic crystal structure (2.2A resolution, R = 22, R(free) = 28) is composed of layers of hydrophobin molecules where the hydrophobic surface areas of the molecules are aligned within the layer. Viewed perpendicular to the aligned hydrophobic surface areas, the molecules in the layer pack together to form six-membered rings, thus leaving small pores in the layer. Similar packing has been observed in the atomic force microscopy images of the self-assembled layers of class II hydrophobin, indicating that the crystal structure resembles that of natural hydrophobin film. The orthorhombic crystal structure (1.0 A resolution, R = 13, R(free) = 15) is composed of fiber-like arrays of protein molecules. Rodlet structures have been observed on amphiphilic layers formed by class I hydrophobins; fibrils of class II hydrophobins appear by vigorous shaking. We propose that the structure of the fibrils and/or rodlets is similar to that observed in the crystal structure.     

MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection-related fungal development, and is required for pathogenicity of M. grisea.     

Structural hierarchy in molecular films of two class II hydrophobins

Hydrophobins are highly surface-active proteins that are specific to filamentous fungi. They function as coatings on various fungal structures, enable aerial growth of hyphae, and facilitate attachment to surfaces. Little is known about their structures and structure-function relationships. In this work we show highly organized surface layers of hydrophobins, representing the most detailed structural study of hydrophobin films so far. Langmuir-Blodgett films of class II hydrophobins HFBI and HFBII from Trichoderma reesei were prepared and analyzed by atomic force microscopy. The films showed highly ordered two-dimensional crystalline structures. By combining our recent results on small-angle X-ray scattering of hydrophobin solutions, we found that the unit cells in the films have dimensions similar to those of tetrameric aggregates found in solutions. Further analysis leads to a model in which the building blocks of the two-dimensional crystals are shape-persistent supramolecules consisting of four hydrophobin molecules. The results also indicate functional and structural differences between HFBI and HFBII that help to explain differences in their properties. The possibility that the highly organized surface assemblies of hydrophobins could allow a route for manufacturing functional surfaces is suggested.     

Self-assembled hydrophobin protein films at the air-water interface: structural analysis and molecular engineering

Hydrophobins are amphiphilic proteins produced by filamentous fungi. They function in a variety of roles that involve interfacial interactions, as in growth through the air-water interface, adhesion to surfaces, and formation of coatings on various fungal structures. In this work, we have studied the formation of films of the class II hydrophobin HFBI from Trichoderma reesei at the air-water interface. Analysis of hydrophobin aqueous solution drops showed that a protein film is formed at the air-water interface. This elastic film was clearly visible, and it appeared to cause the drops to take unusual shapes. Because adhesion and formation of coatings are important biological functions for hydrophobins, a closer structural analysis of the film was made. The method involved picking up the surface film onto a solid substrate and imaging the surface by atomic force microscopy. High-resolution images were obtained showing both the hydrophilic and hydrophobic sides of the film at nanometer resolution. It was found that the hydrophobin film had a highly ordered structure. To study the orientation of molecules and to obtain further insight in film formation, we made variants of HFBI that could be site specifically conjugated. We then used the avidin-biotin interaction as a probe. On the basis of this work, we suggest that the unusual interfacial properties of this type of hydrophobins are due to specific molecular interactions which lead to an ordered network of proteins in the surface films that have a thickness of only one molecule. The interactions between the proteins in the network are likely to be responsible for the unusual surface elasticity of the hydrophobin film.     

Behavior of Trichoderma reesei hydrophobins in solution: interactions, dynamics, and multimer formation

Filamentous fungi utilize small amphiphilic proteins called hydrophobins in their adaptation to the environment. The hydrophobins are used to form coatings on various fungal structures, lower the surface tension of water, and to mediate surface attachment. Hydrophobins function through self-assembly at interfaces, for example, at the air-water interface, and at fungal cellular structures. Despite their high tendency to self assemble at interfaces, hydrophobins can be very soluble in water. To understand the mechanism of hydrophobin self-assembly, in this work, we have studied the behavior of two Trichoderma reesei hydrophobins, HFBI and HFBII in aqueous solution. The main methods used were F?rster resonance energy transfer (FRET) and size exclusion chromatography. A genetically engineered HFBI variant, NCys-HFBI, was utilized for the site-specific labeling of dyes for the FRET experiments. We observed the multimerization of HFBI in a concentration-dependent manner. A change from monomers to tetramers was seen when the hydrophobin concentration was increased. Interaction studies between HFBI and HFBII suggested that at low concentrations homodimers are preferred, and at higher concentrations, the heterotetramers of HFBI and HFBII are formed. In conclusion, the results support the model where hydrophobins in aqueous solutions form multimers by hydrophobic interactions. In contrast to micelles formed by detergents, the hydrophobin multimers are defined in size and involve specific protein-protein interactions.     

Constitutive expression of hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris and its pre‐purification by foam separation during cultivation

Hydrophobins are small surface‐active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor‐made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3‐phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha‐factor prepro‐peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre‐purification using foam separation.     

Complementation of the mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins.

The functional relationship between fungal hydrophobins was studied by complementation analysis of an mpg1(-) gene disruption mutant in Magnaporthe grisea. MPG1 encodes a hydrophobin required for full pathogenicity of the fungus, efficient elaboration of its infection structures and conidial rodlet protein production. Seven heterologous hydrophobin genes were selected which play distinct roles in conidiogenesis, fruit body development, aerial hyphae formation and infection structure elaboration in diverse fungal species. Each hydrophobin was introduced into an mpg1(-) mutant by transformation. Only one hydrophobin gene, SC1 from Schizophyllum commune, was able partially to complement mpg1(-) mutant phenotypes when regulated by its own promoter. In contrast, six of the transformants expressing hydrophobin genes controlled by the MPG1 promoter (SC1 and SC4 from S.commune, rodA and dewA from Aspergillus nidulans, EAS from Neurospora crassa and ssgA from Metarhizium anisopliae) could partially complement each of the diverse functions of MPG1. Complementation was always associated with partial restoration of a rodlet protein layer, characteristic of the particular hydrophobin being expressed, and with hydrophobin surface assembly during infection structure formation. This provides the first genetic evidence that diverse hydrophobin-encoding genes encode functionally related proteins and suggests that, although very diverse in amino acid sequence, the hydrophobins constitute a closely related group of morphogenetic proteins.     

Surface hydrophobicity of Aspergillus nidulans conidiospores and its role in pellet formation

Formation of pellets by Aspergillus nidulans is primarily due to agglomeration of the fungal conidiospores. Although agglomeration of conidiospores has been known for a long time, its mechanism has not been clearly elucidated. To study the influence of the fungal conidiospore wall hydrophobicity on conidiospore agglomeration, pellet formation of an A. nidulans wild type and strains deleted in the conidiospore-wall-associated hydrophobins DewA and RodA was compared at different pH values. From contact angle measurements, RodA was found to be more important for the surface hydrophobicity than DewA. The absence of either hydrophobin led to a decrease in the relative amount of biomass present as pellets at all pH values as well as a decrease in the average size of the pellets. For all strains, an increasing alkalinity of the medium resulted in an increased pellet formation. Together with measurements of electrophoretic mobility, it is concluded that both the electrical charge and hydrophobicity of the conidiospores affects the pellet formation but that the conidiospore agglomeration process cannot be ascribed to these factors alone.     

Surface modifications created by using engineered hydrophobins

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.     

Recruitment of class I hydrophobins to the air:water interface initiates a multi-step process of functional amyloid formation

Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.     

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.     

The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity

Class I hydrophobins are fungal proteins that self-assemble into robust amphipathic rodlet monolayers on the surface of aerial structures such as spores and fruiting bodies. These layers share many structural characteristics with amyloid fibrils and belong to the growing family of functional amyloid-like materials produced by microorganisms. Although the three-dimensional structure of the soluble monomeric form of a class I hydrophobin has been determined, little is known about the molecular structure of the rodlets or their assembly mechanism. Several models have been proposed, some of which suggest that the Cys3-Cys4 loop has a critical role in the initiation of assembly or in the polymeric structure. In order to provide insight into the relationship between hydrophobin sequence and rodlet assembly, we investigated the role of the Cys3-Cys4 loop in EAS, a class I hydrophobin from Neurospora crassa. Remarkably, deletion of up to 15 residues from this 25-residue loop does not impair rodlet formation or reduce the surface activity of the protein, and the physicochemical properties of rodlets formed by this mutant are indistinguishable from those of its full-length counterpart. In addition, the core structure of the truncation mutant is essentially unchanged. Molecular dynamics simulations carried out on the full-length protein and this truncation mutant binding to an air-water interface show that, although it is hydrophobic, the loop does not play a role in positioning the protein at the surface. These results demonstrate that the Cys3-Cys4 loop does not have an integral role in the formation or structure of the rodlets and that the major determinant of the unique properties of these proteins is the amphipathic core structure, which is likely to be preserved in all hydrophobins despite the high degree of sequence variation across the family.     

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k_cat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.     

Probing Structural Changes during Self-assembly of Surface-Active Hydrophobin Proteins that Form Functional Amyloids in Fungi

Hydrophobins are amphiphilic proteins secreted by filamentous fungi in a soluble form, which can self-assemble at hydrophilic/hydrophobic or water/air interfaces to form amphiphilic layers that have multiple biological roles. We have investigated the conformational changes that occur upon self-assembly of six hydrophobins that form functional amyloid fibrils with a rodlet morphology. These hydrophobins are present in the cell wall of spores from different fungal species. From available structures and NMR chemical shifts, we established the secondary structures of the monomeric forms of these proteins and monitored their conformational changes upon amyloid rodlet formation or thermal transitions using synchrotron radiation circular dichroism and Fourier-transform infrared spectroscopy (FT-IR). Thermal transitions were followed by synchrotron radiation circular dichroism in quartz cells that allowed for microbubbles and hence water/air interfaces to form and showed irreversible conformations that differed from the rodlet state for most of the proteins. In contrast, thermal transitions on hermetic calcium fluoride cells showed reversible conformational changes. Heating hydrophobin solutions with a water/air interface on a silicon crystal surface in FT-IR experiments resulted in a gain in β-sheet content typical of amyloid fibrils for all except one protein. Rodlet formation was further confirmed by electron microscopy. FT-IR spectra of pre-formed hydrophobin rodlet preparations also showed a gain in β-sheet characteristic of the amyloid cross-β structure. Our results indicate that hydrophobins are capable of significant conformational plasticity and the nature of the assemblies formed by these surface-active proteins is highly dependent on the interface at which self-assembly takes place.     

Surface Modifications Created by Using Engineered Hydrophobins

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.     

Two crystal structures of Trichoderma reesei hydrophobin HFBI--the structure of a protein amphiphile with and without detergent interaction

Hydrophobins are small fungal proteins that are highly surface active and possess a unique ability to form amphiphilic membranes through spontaneous self-assembly. The first crystal structure of a hydrophobin, Trichoderma reesei HFBII, revealed the structural basis for the function of this amphiphilic protein--a patch consisting of hydrophobic side chains on the protein surface. Here, the crystal structures of a native and a variant T. reesei hydrophobin HFBI are presented, revealing the same overall structure and functional hydrophobic patch as in the HFBII structure. However, some structural flexibility was found in the native HFBI structure: The asymmetric unit contained four molecules, and, in two of these, an area of seven residues was displaced as compared to the two other HFBI molecules and the previously determined HFBII structure. This structural change is most probably induced by multimer formation. Both the native and the N-Cys-variant of HFBI were crystallized in the presence of detergents, but an association between the protein and a detergent was only detected in the variant structure. There, the molecules were arranged into an extraordinary detergent-associated octamer and the solvent content of the crystals was 75%. This study highlights the conservation of the fold of class II hydrophobins in spite of the low sequence identity and supports our previous suggestion that concealment of the hydrophobic surface areas of the protein is the driving force in the formation of multimers and monolayers in the self-assembly process.