Hydrophobin HFBI Research Articles

Novel Hydrophobin Fusion Tags for Plant-Produced Fusion Proteins.

Hydrophobin fusion technology has been applied in the expression of several recombinant proteins in plants. Until now, the technology has relied exclusively on the Trichoderma reesei hydrophobin HFBI. We screened eight novel hydrophobin tags, T. reesei HFBII, HFBIII, HFBIV, HFBV, HFBVI and Fusarium verticillioides derived HYD3, HYD4 and HYD5, for production of fusion proteins in plants and purification by two-phase separation. To study the properties of the hydrophobins, we used N-terminal and C-terminal GFP as a fusion partner. Transient expression of the hydrophobin fusions in Nicotiana benthamiana revealed large variability in accumulation levels, which was also reflected in formation of protein bodies. In two-phase separations, only HFBII and HFBIV were able to concentrate GFP into the surfactant phase from a plant extract. The separation efficiency of both tags was comparable to HFBI. When the accumulation was tested side by side, HFBII-GFP gave a better yield than HFBI-GFP, while the yield of HFBIV-GFP remained lower. Thus we present here two alternatives for HFBI as functional fusion tags for plant-based protein production and first step purification.

Self-Assembly and Conformational Changes of Hydrophobin Classes at the Air-Water Interface.

We use surface-specific vibrational sum-frequency generation spectroscopy (VSFG) to study the structure and self-assembling mechanism of the class I hydrophobin SC3 from Schizophyllum commune and the class II hydrophobin HFBI from Trichoderma reesei. We find that both hydrophobins readily accumulate at the water-air interface and form rigid, highly ordered protein films that give rise to prominent VSFG signals. We identify several resonances that are associated with β-sheet structures and assign them to the central β-barrel core present in both proteins. Differences between the hydrophobin classes are observed in their interfacial self-assembly. For HFBI, we observe no changes in conformation upon adsorption to the water surface. For SC3, we observe an increase in β-sheet-specific signals that supports a surface-driven self-assembly mechanism in which the central β-barrel remains intact and stacks into a larger-scale architecture, amyloid-like rodlets.

Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System.

Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution.

Mechanism of Nonpolar Model Substances to Inhibit Primary Gushing Induced by Hydrophobin HFBI.

In this work, the interactions of a well-studied hydrophobin with different types of nonpolar model substances and their impact on primary gushing is evaluated. The nature, length, and degree of saturation of nonpolar molecules are key parameters defining the gushing ability or inhibition. When mixed with hydrophobins, the nonpolar molecule-hydrophobin assembly acts as a less gushing or no gushing system. This effect can be explained in the framework of a competition effect between non-polar systems and CO2 to interact with the hydrophobic patch of the hydrophobin. Interactions of these molecules with hydrophobins are promoted as a result of the similar size of the nonpolar molecules with the hydrophobic patch of the protein, at the expense of the formation of nanobubbles with CO2. In order to prove the presence of interactions and to unravel the mechanisms behind them, a complete set of experimental techniques was used. Surface sensitive techniques clearly show the presence of the interactions, whose nature is not covalent nor hydrogen bonding according to infrared spectroscopy results. Interactions were also reflected by particle size analysis in which mixtures of particles displayed larger size than their pure component counterparts. Upon mixing with nonpolar molecules, the gushing ability of the protein is significantly disrupted.

Charge-based engineering of hydrophobin HFBI: effect on interfacial assembly and interactions.

Hydrophobins are extracellular proteins produced by filamentous fungi. They show a variety of functions at interfaces that help fungi to adapt to their environment by, for example, adhesion, formation of coatings, and lowering the surface tension of water. Hydrophobins fold into a globular structure and have a distinct hydrophobic patch on their surface that makes these proteins amphiphilic. Their amphiphilicity implies interfacial assembly, but observations indicate that intermolecular interactions also contribute to their functional properties. Here, we used the class II hydrophobin HFBI from Trichoderma reesei as a model to understand the structural basis for the function of hydrophobins. Four different variants were made in which charged residues were mutated. The residues were chosen to probe the role of different regions of the hydrophilic part of the proteins. Effects of the mutations were studied by analyzing the formation and structure of self-assembled layers, multimerization in solution, surface adhesion, binding of secondary layers of proteins on hydrophobins, and the viscoelastic behavior of the air-water interface during formation of protein films; the comparison showed clear differences between variants only in the last two analyses. Surface viscoelasticity behavior suggests that the formation of surface layers is regulated by specific interactions that lead to docking of proteins to each other. One set of mutations led to assemblies with a remarkably high elasticity at the air-water interface (1.44 N/m). The variation of binding of secondary layers of protein on surface-adsorbed hydrophobins suggest a mechanism for a proposed function of hydrophobins, namely, that hydrophobins can act as a specific adhesive layer for the binding of macromolecules to interfaces.

Suspended crystalline films of protein hydrophobin I (HFBI)

Protein interfaces play an essential role in both natural and man-made materials as stabilizers, sensors, catalysts, and selective channels for ions and small molecules. Probing the molecular arrangement within such interfaces is of prime importance to understand the relation between structure and functionality. Here we report on the preparation and characterization of large area suspended crystalline films of class II hydrophobin HFBI. This small, amphiphilic globular protein readily self-assembles at the air–water interface into a 2D hexagonal lattice which can be transferred onto a holey carbon electron microscopy grid yielding large areas of hundreds of square micrometers intact hydrophobin film spun across micron-sized holes. Fourier transform analysis of low-dose electron microscopy images and selected area electron diffraction profiles reveal a unit cell dimension a=5.6±0.1nm, in agreement with reported atomic force microscopy studies on solid substrates and grazing incidence X-ray scattering experiments at the air–water interface. These findings constitute the first step towards the utilization of large-area suspended crystalline hydrophobin films as membranes for ultrapurification and chiral separation or as biological substrates for ultrafast electron diffraction.

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.

Effect of a magnetic field on dispersion of a hop extract and the influence on gushing of beer

Hydrophobins are surface active molecules that cause gushing of beer. Gushing is vigorous overfoaming of carbonated beverages without any shaking. A hop extract was used to decrease gushing of wort induced by hydrophobin HFBI. The influence of a magnetic field on dispersion of the hop extract was used to decrease gushing by HFBI. The results indicate that when a magnetic field exerted on hop extract, this compound is dispersed more and smaller particles are formed. Therefore, the specific surface areas of the particles are increased and interact with larger numbers of hydrophobins. This resulted in less gushing by HFBI. When hydrophobins and hop extract together were submitted to magnetic field more gushing was obtained than in the absence of magnet. This is due to the extensive dispersion of the combination by the magnet and can be limited by using less amount of hop extract.

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.

Molecular simulation of adsorption of hydrophobin HFBI to the air–water, DPPC–water and decane–water interfaces

Molecular dynamics simulation has been used to model the adsorption of the class II hydrophibin HFBI from Trichoderma reesei at the vacuum–water, DPPC bilayer–water and decane–water interfaces. When two HFBI molecules were simulated in a water-box they self-associated into a dimer with the two hydrophobic patches of the molecule forming the dimer interface. When at the fluid interfaces HFBI adsorbed to both the vacuum–water and decane–water interfaces by adopting a conformation where the hydrophobic patch is parallel to and embedded into the interface. At the DPPC bilayer interface, however, the hydrophobic patch is oriented perpendicular to the interface, although still partially embedded into the bilayer. This difference in adsorbed conformation orientation is most likely due to the presence of the hydrophilic phosphatidyl head groups that are found preferentially at the surface of the bilayer. The degree of tertiary conformational change in the adsorbed HFBI molecules is low at the vacuum–water, slightly greater at the DPPC–water interface, and quite substantial at the decane–water interface. Changes in secondary structure are small, with only HFBI at the decane–water interface showing a significant reduction in beta structure. The effect of the HFBI molecule on the DPPC bilayer is marked, with changes in the ordering of the DPPC molecules being observed not only on the side of the bilayer where the protein is adsorbed, but also on the side opposite to that where the protein adsorbs. The results support the view that hydrophobin molecules behave as rigid janus particles when they adsorb to fluid interfaces.

Solid-support immobilization of a “swing” fusion protein for enhanced glucose oxidase catalytic activity

The strategic surface immobilization of a protein can add new functionality to a solid substrate; however, protein activity, e.g., enzymatic activity, can be drastically decreased on immobilization onto a solid surface. The concept of a designed and optimized “molecular interface” is herein introduced in order to address this problem. In this study, molecular interface was designed and constructed with the aim of attaining high enzymatic activity of a solid-surface-immobilized a using the hydrophobin HFBI protein in conjunction with a fusion protein of HFBI attached to glucose oxidase (GOx). The ability of HFBI to form a self-organized membrane on a solid surface in addition to its adhesion properties makes it an ideal candidate for immobilization. The developed fusion protein was also able to form an organized membrane, and its structure and immobilized state on a solid surface were investigated using QCM-D measurements. This method of immobilization showed retention of high enzymatic activity and the ability to control the density of the immobilized enzyme. In this study, we demonstrated the importance of the design and construction of molecular interface for numerous purposes. This method of protein immobilization could be utilized for preparation of high throughput products requiring structurally ordered molecular interfaces, in addition to many other applications.

Kinetic and Equilibrium Aspects of Adsorption and Desorption of Class II Hydrophobins HFBI and HFBII at Silicon Oxynitride/Water and Air/Water Interfaces

Hydrophobins are relatively small globular proteins produced by filamentous fungi. They display unusual high surface activity and are implied as mediators of attachment to surfaces, which has resulted in high scientific and technological interest. In this work we focus on kinetic and equilibrium aspects of adsorption and desorption properties of two representatives of class II hydrophobins, namely HFBI and HFBII, at a negatively charged hydrophilic solid/water interface and at the air/water interface. The layers formed at the air/liquid interface were examined in a Langmuir trough, whereas layers formed at the solid/liquid interface were studied using dual polarization interferometry (DPI) under different flow conditions. For comparison, another globular protein, lysozyme, was also investigated. It was found that both the adsorbed amount and the adsorption kinetics were different for HFBI and HFBII, and the adsorption behavior of both hydrophobins on the negatively charged surface displayed some unusual features. For instance, even though the adsorption rate for HFBI was slowed down with increasing adsorbed amount as expected from packing constraints at the interface, the adsorption kinetics curves for HFBII displayed a region indicating adsorption cooperativity. Further, it was found that hydrophobin layers formed under flow partly desorbed when the flow was stopped, and the desorption rate for HFBII was enhanced in the presence of hydrophobins in solution.

Modification of interfacial forces by hydrophobin HFBI

Using a surface force balance, we have measured the forces between bare (hydrophilic) mica surfaces, and between hydrophobized mica surfaces, in each case coated with the amphiphilic protein hydrophobin (HFBI) from Trichoderma reesei. We additionally characterized these surfaces by contact angle measurements and AFM. The results are consistent with the formation of hydrophobic surfaces exposed by HFBI adsorbed on the hydrophilic substrate, and hydrophilic surfaces exposed by HFBI adsorbed on the hydrophobic substrate. In particular, friction between HFBI surfaces on hydrophobized mica, exposing hydrophilic surfaces, is an order of magnitude lower than friction between HFBI-coated hydrophilic surfaces, which expose the hydrophobic side of the protein to the water interface. This result can be readily understood in terms of the greater hydration level of the exposed outer surfaces in the former case.

Hydrophobin: fluorosurfactant-like properties without fluorine

The stabilization of fluorous oil droplets in aqueous environment is a critical issue in the preparation of emulsified systems for biomedical applications and in emulsion polymerization technology, due to the extreme immiscibility of aqueous and fluorous phases. We present here a detailed study on the behavior of the hydrophobin HFBI, i.e. a small natural protein endowed with exceptional surface activity, at the interface between aqueous and fluorous phases. HFBI behaves as an efficient and sustainable surfactant at remarkably low concentrations and forms a strong and elastic film at the interface between the two phases. We also show proof-of-concept experiments on the use of HFBI as a surfactant in fluorous oil/water emulsified systems and in microfluidic circuits.

Self-assembly of Class II Hydrophobins on Polar Surfaces

Hydrophobins are structural proteins produced by filamentous fungi that are amphiphilic and function through self-assembling into structures such as membranes. They have diverse roles in the growth and development of fungi, for example in adhesion to substrates, for reducing surface tension to allow aerial growth, in forming protective coatings on spores and other structures. Hydrophobin membranes at the air-water interface and on hydrophobic solids are well studied, but understanding how hydrophobins can bind to a polar surface to make it more hydrophobic has remained unresolved. Here we have studied different class II hydrophobins for their ability to bind to polar surfaces that were immersed in buffer solution. We show here that the binding under some conditions results in a significant increase of water contact angle (WCA) on some surfaces. The highest contact angles were obtained on cationic surfaces where the hydrophobin HFBI has an average WCA of 62.6° at pH 9.0, HFBII an average of 69.0° at pH 8.0, and HFBIII had an average WCA of 61.9° at pH 8.0. The binding of the hydrophobins to the positively charged surface was shown to depend on both pH and ionic strength. The results are significant for understanding the mechanism for formation of structures such as the surface of mycelia or fungal spore coatings as well as for possible technical applications.

Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry

The class II hydrophobin HFBI from Trichoderma reesei was heterologously expressed by Pichia pastoris using pPIC9 vector under the control of the promoter AOX1. The recombinant HFBI (rHFBI) was purified by ultrafiltration and reverse-phase high performance liquid chromatography. Tricine-SDS-PAGE and Western blotting demonstrated that rHFBI with the expected molecular weight of 7.5 kDa was secreted into the culture medium. X-ray photoelectron spectroscopy and water contact angle measurements indicated that rHFBI could lead to the conversion of the wettability of the hydrophobic siliconized glass and hydrophilic mica surfaces relying on the self-assembly membrane on hydrophobic/hydrophilic interfaces. It was demonstrated that rHFBI had the ability to stabilize oil droplets, which was far excess of the class I hydrophobin HGFI heterologously expressed in P. pastoris (rHGFI) and the typical food emulsifier sodium caseinate. In gushing experiments, it was shown that rHFBI was a strong gushing inducer in beer, whereas rHGFI did not display any signs of gushing. This provided the potential of rHFBI to be used as a novel emulsifying agent and a predictor of gushing risk.

Dynamic Light Scattering (DLS) as a Tool to Detect CO2-Hydrophobin Structures and Study the Primary Gushing Potential of Beer

When weather conditions are damp, barley crops can be infected by fungi that produce amphiphilic proteins called hydrophobins. We postulated that these hydrophobins contaminate CO2 bubbles during beer carbonation or shaking. These molecules prevent the dissolution or redissolution of CO2 and, thus, stabilize CO2 bubbles. These bubbles contain energy (overpressure) that is released when the beverage container is opened, causing the simultaneous nucleation of many bubbles, which leads to primary gushing. In this paper, we present validation of our hypothesis and show that dynamic light scattering (DLS) can be used as a tool to study the primary gushing phenomenon. The results indicate that both hydrophobins and CO2 are necessary to induce gushing. Indeed, addition of a crude mycelium extract containing hydrophobin HFBI to different beverages such as beer, soda, and sparkling water led to gushing, whereas addition to still mineral water did not. Comparison of a commercial gushing and nongushing beer using DLS revealed the presence of particles with a diameter of approx. 100 nm in the gushing beer. According to our hypothesis, these 100-nm particles should be nanobubbles stabilized by hydrophobin assemblages. The presence of these particles was also observed in all gushing beverages obtained after addition of the hydrophobin-containing mycelium extract.

Polysaccharides as Precipitants in Protein Crystallization for X-Ray Diffraction Studies

A novel polysaccharide-based method for protein crystallization has been developed for industrial purposes. In this paper, we describe the application of this new method in crystal growth for X-ray diffraction structural studies of proteins. We have tested the crystallization of three commercially available proteins—lysozyme, xylose isomerase, and xylanase—and of two research targets—the amphiphilic protein hydrophobins HFBI and HFBII—on the polysaccharides alginate, chitosan, pectin, and dextrin, and enzymatic hydrolysates of alginate and pectin. With the use of polysaccharides, we managed to grow crystals of each protein. Crystals of lysozyme, xylose isomerase, and xylanase were suitable for X-ray diffraction studies on the basis of size, shape, and diffraction tests. Data were collected on xylanase crystals, and the structure was refined to ensure that the polysaccharide has no effect on structure determination. Both hydrophobins also yielded crystals suitable for X-ray analysis, once a suitable additive was used in combination with the polysaccharide. Thus, we conclude that this novel method is applicable also in protein structural studies. We suggest that the underlying phenomena in crystallization with polysaccharides is the formation of a polysaccharide−protein phase where the phase boundary acts as the initiation point for crystal formation.

Noncovalently functionalized multi-wall carbon nanotubes in aqueous solution using the hydrophobin HFBI and their electroanalytical application

A novel noncovalent approach was developed for the functionalization of multi-wall carbon nanotubes (MWNTs) using the hydrophobin, HFBI. Owing to the amphipathic nature, HFBI can be adopted onto the surface of MWNTs to form HFBI–MWNTs nanocomposite with good dispersion in water. The HFBI–MWNTs nanocomposite was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and water contact angle measurements (WCA). Furthermore, a glucose biosensor was developed based on HFBI–MWNTs by a one-step casting method. The resulting biosensor displayed high sensitivity, wider linear range, low detection limit, and fast response for glucose detection, which implicated that the HFBI–MWNTs nanocomposite film holds great promise in the design of electrochemical devices, such as sensors and biosensors.

Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana.

Insufficient accumulation levels of recombinant proteins in plants and the lack of efficient purification methods for recovering these valuable proteins have hindered the development of plant biotechnology applications. Hydrophobins are small and surface-active proteins derived from filamentous fungi that can be easily purified by a surfactant-based aqueous two-phase system. In this study, the hydrophobin HFBI sequence from Trichoderma reesei was fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants by Agrobacterium tumefaciens infiltration. The HFBI fusion significantly enhanced the accumulation of GFP, with the concentration of the fusion protein reaching 51% of total soluble protein, while also delaying necrosis of the infiltrated leaves. Furthermore, the endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of large novel protein bodies. A simple and scalable surfactant-based aqueous two-phase system was optimized to recover the HFBI fusion proteins from leaf extracts. The single-step phase separation was able to selectively recover up to 91% of the GFP-HFBI up to concentrations of 10 mg mL(-1). HFBI fusions increased the expression levels of plant-made recombinant proteins while also providing a simple means for their subsequent purification. This hydrophobin fusion technology, when combined with the speed and posttranslational modification capabilities of plants, enhances the value of transient plant-based expression systems.