Aggregation and self-assembly of hydrophobins from Trichoderma reesei: low-resolution structural models

Hydrophobins are secreted fungal proteins, which have diverse roles in fungal growth and development. They lower the surface tension of water, work as adhesive agents and coatings, and function through self-assembly. One of the characteristic properties of hydrophobins is their tendency to form fibrillar or rod-like aggregates at interfaces. Their structure is still poorly known. In a step to elucidate the structure/function relation of hydrophobin self-assembly, we present the low-resolution structure of self-assembled fibrils of the class II hydrophobin HFBII from Trichoderma reesei based on small and wide-angle x-ray scattering. We first studied the solution state (10 mg/mL) of both HFBI and HFBII and showed that they formed assemblages in aqueous solution, which have a radius of gyration of ~24 A and maximum dimension of ~65 A, corresponding to the size of a tetramer. This result was supported by size-exclusion chromatography. Undried samples of HFBII fibrils had a monoclinic crystalline structure, which changed to hexagonal when the material was dried. A low-resolution structure for the HFBII fibrils is suggested. There are data in the literature based on staining properties suggesting that hydrophobins of class I form assemblies with an amyloid structure. Comparison of the HFBII data (x-ray results, staining with thioflavin T) to published data showed that the HFBII assemblages are not amyloid.     

Interaction and comparison of a class I hydrophobin from Schizophyllum commune and class II hydrophobins from Trichoderma reesei

Hydrophobins fulfill a wide spectrum of functions in fungal growth and development. These proteins self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. Hydrophobins are divided into two classes based on their hydropathy patterns and solubility. We show here that the properties of the class II hydrophobins HFBI and HFBII of Trichoderma reesei differ from those of the class I hydrophobin SC3 of Schizophyllum commune. In contrast to SC3, self-assembly of HFBI and HFBII at the water-air interface was neither accompanied by a change in secondary structure nor by a change in ultrastructure. Moreover, maximal lowering of the water surface tension was obtained instantly or took several minutes in the case of HFBII and HFBI, respectively. In contrast, it took several hours in the case of SC3. Oil emulsions prepared with HFBI and SC3 were more stable than those of HFBII, and HFBI and SC3 also interacted more strongly with the hydrophobic Teflon surface making it wettable. Yet, the HFBI coating did not resist treatment with hot detergent, while that of SC3 remained unaffected. Interaction of all the hydrophobins with Teflon was accompanied with a change in the circular dichroism spectra, indicating the formation of an alpha-helical structure. HFBI and HFBII did not affect self-assembly of the class I hydrophobin SC3 of S. commune and vice versa. However, precipitation of SC3 was reduced by the class II hydrophobins, indicating interaction between the assemblies of both classes of hydrophobins.     

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.     

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.     

Interactions of hydrophobin proteins in solution studied by small-angle X-ray scattering

Hydrophobins are a group of very surface-active, fungal proteins known to self-assemble on various hydrophobic/hydrophilic interfaces. The self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins are also soluble in water. Here, the association of hydrophobins HFBI and HFBII from Trichoderma reesei in aqueous solution was studied using small-angle x-ray scattering. Both HFBI and HFBII exist mainly as tetramers in solution in the concentration range 0.5-10 mg/ml. The assemblies of HFBII dissociate more easily than those of HFBI, which can tolerate changes of pH from 3 to 9 and temperatures in the range 5°C-60°C. The self-association of HFBI and HFBII is mainly driven by the hydrophobic effect, and addition of salts along the Hofmeister series promotes the formation of larger assemblies, whereas ethanol breaks the tetramers into monomers. The possibility that the oligomers in solution form the building blocks of the self-assembled film at the air/water interface is discussed.     

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.     

Hydrophobins, from molecular structure to multiple functions in fungal development

Mycelial fungi secrete small, cysteine-rich, proteins, called hydrophobins, that self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes, highly insoluble in case of Class I hydrophobins. By self-assembly at the culture medium-air interface they greatly lower the surface tension enabling emergent structures to grow into the air. By self-assembly at the interface between the hydrophilic cell wall and the air or any other hydrophobic environment, these emergent structures are coated with a hydrophobin membrane. These properties allow hydrophobins to fulfil a broad spectrum of functions in fungal development. They are involved in formation of aerial (reproductive) structures, in aerial dispersion of spores, and they line air channels within fruiting bodies with a hydrophobic coating, probably serving gas exchange. Hydrophobins also mediate hyphal attachment to hydrophobic surfaces such as those of plants. Moreover, they appear involved in complex interhyphal interactions, and in interactions with algae in lichens. Their resistance towards chemical and enzymatic treatments suggests that assembled hydrophobins also protect fungal emergent structures against adverse environmental conditions.     

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.     

Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile

Hydrophobins are proteins specific to filamentous fungi. Hydrophobins have several important roles in fungal physiology, for example, adhesion, formation of protective surface coatings, and the reduction of the surface tension of water, which allows growth of aerial structures. Hydrophobins show remarkable biophysical properties, for example, they are the most powerful surface-active proteins known. To this point the molecular basis of the function of this group of proteins has been largely unknown. We have now determined the crystal structure of the hydrophobin HFBII from Trichoderma reesei at 1.0 A resolution. HFBII has a novel, compact single domain structure containing one alpha-helix and four antiparallel beta-strands that completely envelop two disulfide bridges. The protein surface is mainly hydrophilic, but two beta-hairpin loops contain several conserved aliphatic side chains that form a flat hydrophobic patch that makes the molecule amphiphilic. The amphiphilicity of the HFBII molecule is expected to be a source for surface activity, and we suggest that the behavior of this surfactant is greatly enhanced by the self-assembly that is favored by the combination of size and rigidity. This mechanism of function is supported by atomic force micrographs that show highly ordered arrays of HFBII at the air water interface. The data presented show that much of the current views on structure function relations in hydrophobins must be re-evaluated.     

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.     

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.     

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.     

Hydrophobin Film Structure for HFBI and HFBII and Mechanism for Accelerated Film Formation

Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology.     

Spontaneous self-assembly of SC3 hydrophobins into nanorods in aqueous solution

Hydrophobins are small surface active proteins secreted by filamentous fungi. Because of their ability to self-assemble at hydrophilic-hydrophobic interfaces, hydrophobins play a key role in fungal growth and development. In the present work, the organization in aqueous solution of SC3 hydrophobins from the fungus Schizophyllum commune was assessed using Dynamic Light Scattering, Atomic Force Microscopy and fluorescence spectroscopy. These complementary approaches have demonstrated that SC3 hydrophobins are able not only to spontaneously self-assemble at the air-water interface but also in pure water. AFM experiments evidenced that hydrophobins self-assemble in solution into nanorods. Fluorescence assays with thioflavin T allowed establishing that the mechanism governing SC3 hydrophobin self-assembly into nanorods involves β-sheet stacking. SC3 assembly was shown to be strongly influenced by ionic strength and solution pH. The presence of a very low ionic strength significantly favoured the protein self-assembly but a further increase of ions in solution disrupted the protein assembly. It was assessed that solution pH had a significant effect on the SC3 hydrophobins organization. In peculiar, the self-assembly process was considerably reduced at acidic pH. Our findings demonstrate that the self-assembly of SC3 hydrophobins into nanorods of well-defined length can be directly controlled in solution. Such control allows opening the way for the development of new smart self-assembled structures for targeted applications.     

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.     

Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei

Hydrophobins are surface-active proteins produced by filamentous fungi, where they seem to be ubiquitous. They have a variety of roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension. Hydrophobins can be divided into two classes based on the hydropathy profile of their primary sequence. We have studied the adhesion behavior of two Trichoderma reesei class II hydrophobins, HFBI and HFBII, as isolated proteins and as fusion proteins. Both hydrophobins were produced as C-terminal fusions to the core of the hydrolytic enzyme endoglucanase I from the same organism. It was shown that as a fusion partner, HFBI causes the fusion protein to efficiently immobilize to hydrophobic surfaces, such as silanized glass and Teflon. The properties of the surface-bound protein were analyzed by the enzymatic activity of the endoglucanase domain, by surface plasmon resonance (Biacore), and by a quartz crystal microbalance. We found that the HFBI fusion forms a tightly bound, rigid surface layer on a hydrophobic support. The HFBI domain also causes the fusion protein to polymerize in solution, possibly to a decamer. Although isolated HFBII binds efficiently to surfaces, it does not cause immobilization as a fusion partner, nor does it cause polymerization of the fusion protein in solution. The findings give new information on how hydrophobins function and how they can be used to immobilize fusion proteins.     

The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei

Hydrophobins are small fungal surface active proteins that self-assemble at interfaces into films with nanoscale structures. The hydrophobin HFBI from Trichoderma reesei has been shown to associate in solution into tetramers but the role of this association on the function of HFBI has remained unclear. We produced two HFBI variants that showed a significant shift in solution association equilibrium towards the tetramer state. However, this enhanced solution association did not alter the surface properties of the variant HFBIs. The results show that there is not a strong relationship between HFBI solution association state and surface properties such as surface activity.     

Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI

Many characteristics of fungal hydrophobins, such as an ability to change hydrophobicity of different surfaces, have potential for several applications. The large-scale processes of production and isolation of these proteins susceptible to aggregation and attachment to interfacial surfaces still needs to be studied. We report for the first time on a method for a gram-scale production and purification of a hydrophobin, HFBI of Trichoderma reesei. A high production level of the class II hydrophobin (0.6 g l(-1)) was obtained by constructing a T. reesei HFBI-overproducing strain containing three copies of the hfb1 gene. The strain was cultivated on glucose-containing medium, which induces expression of hfb1. HFBI hydrophobin was purified from the cell walls of the fungus because most of the HFBI was cell-bound (80%). Purification was carried out with a simple three-step method involving extraction of the mycelium with 1% SDS at pH 9.0, followed by KCl precipitation to remove SDS, and hydrophobic interaction chromatography. The yield was 1.8 g HFBI from mycelium (419 g dw), derived from 15 l of culture. HFBI was shown to be rather unstable to N-terminal asparagine deamidation and also, to some extent, to non-specific proteases although its thermostability was excellent.     

The functional role of Cys3–Cys4 loop in hydrophobin HGFI

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 signal sequence mediates efficient secretion of recombinant proteins in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris is an important eukaryotic organism for the expression, processing, and secretion of recombinant proteins. Here, the secretion of enhanced green fluorescent protein (EGFP) in P. pastoris by using three novel secretion signals originating from the HFBI and HFBII class 2 hydrophobins of Trichoderma reesei was investigated. EGFP was fused to the carboxyl terminus of hydrophobin secretion signals and expressed under the control of the constitutive GAP promoter. In every case, recombinant EGFP entered the secretory pathway of P. pastoris. SDS-polyacrylamide gel electrophoresis, Western blot analysis of the cells' supernatant, and fluorescence measurements on single-cell level via flow cytometry confirmed the efficient secretion of EGFP mediated by the novel secretion sequences. In conclusion, the data clearly show that the secretion sequences derived from HFBI and HFBII of T. reesei have the potential to achieve an efficient secretion of heterologous proteins in P. pastoris. Due to the small size of the hydrophobin-derived secretion signals, their coding sequence can be easily introduced to the gene of interest by PCR.