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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.     

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.     

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.     

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.     

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.     

Self-assembly of proteins into a three-dimensional multilayer system: Investigation of the surface of the human fungal pathogen Aspergillus fumigatus

Hydrophobins are small surface active proteins that fulfil a wide spectrum of functions in fungal growth and development. The human fungal pathogen Aspergillus fumigatus expresses RodA hydrophobins that self-assemble on the outer conidial surface into tightly organized nanorods known as rodlets. AFM investigation of the conidial surface allows us to evidence that RodA hydrophobins self-assemble into rodlets through bilayers. Within bilayers, hydrophilic domains of hydrophobins point inward, thus making a hydrophilic core, while hydrophobic domains point outward. AFM measurements reveal that several rodlet bilayers are present on the conidial surface thus showing that proteins self-assemble into a complex three-dimensional multilayer system. The self-assembly of RodA hydrophobins into rodlets results from attractive interactions between stacked β-sheets, which conduct to a final linear cross-β spine structure. A Monte Carlo simulation shows that anisotropic interactions are the main driving forces leading the hydrophobins to self-assemble into parallel rodlets, which are further structured in nanodomains. Taken together, these findings allow us to propose a mechanism, which conducts RodA hydrophobins to a highly ordered rodlet structure. The mechanism of hydrophobin assembly into rodlets offers new prospects for the development of more efficient strategies leading to disruption of rodlet formation allowing a rapid detection of the fungus by the immune system.     

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.     

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.     

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.     

Bond Strengthening in Oral Bacterial Adhesion to Salivary Conditioning Films

Transition from reversible to irreversible bacterial adhesion is a highly relevant but poorly understood step in initial biofilm formation. We hypothesize that in oral biofilm formation, irreversible adhesion is caused by bond strengthening due to specific bacterial interactions with salivary conditioning films. Here, we compared the initial adhesion of six oral bacterial strains to salivary conditioning films with their adhesion to a bovine serum albumin (BSA) coating and related their adhesion to the strengthening of the binding forces measured with bacteria-coated atomic force microscopy cantilevers. All strains adhered in higher numbers to salivary conditioning films than to BSA coatings, and specific bacterial interactions with salivary conditioning films were accompanied by stronger initial adhesion forces. Bond strengthening occurred on a time scale of several tens of seconds and was slower for actinomyces than for streptococci. Nonspecific interactions between bacteria and BSA coatings strengthened twofold faster than their specific interactions with salivary conditioning films, likely because specific interactions require a closer approach of interacting surfaces with the removal of interfacial water and a more extensive rearrangement of surface structures. After bond strengthening, bacterial adhesion forces with a salivary conditioning film remained stronger than those with BSA coatings.     

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.     

Nanoscale reduction in surface friction of polymer surfaces modified with Sc3 hydrophobin from Schizophyllum commune

Hydrophobins are amphipathic self-assembling proteins secreted by filamentous fungi that exhibit remarkable ability to modify synthetic surfaces. Thin coatings of Sc3 hydrophobin isolated from the wood-rotting fungus Schizophyllum commune were prepared via spin coating and adsorption techniques onto polymeric surfaces. Surface morphology and nanotribological characteristics of the films were evaluated using lateral force microscopy (LFM) and nanoindentation techniques. This paper reports the first observation of reduction in nanoscale relative surface friction of Sc3 hydrophobin protein modified polymeric surfaces. Relative friction coefficients were dramatically reduced and hydrophilicity increased for polymer surfaces modified with Sc3 hydrophobin thin films. Morphology of the protein films as well as degree of surface modification was observed to be a function of film formation technique and composition of the substrate.