Hydrophobins: Proteins that self assemble at interfaces

Current Opinion in Colloid & Interface Science 14 (2009) 356–363

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Current Opinion in Colloid & Interface Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c o c i s

Hydrophobins: Proteins that self assemble at interfaces Markus B. Linder ⁎ VTT Technical Research Centre of Finland, Biotechnology, Tietotie 2, FIN-02044 VTT, Finland

a r t i c l e

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Article history: Received 1 November 2008 Received in revised form 12 March 2009 Accepted 2 April 2009 Available online 8 May 2009 Keywords: Hydrophobin Surface active protein Self assembly

a b s t r a c t Hydrophobins are surface active proteins that are produced by filamentous fungi. They are interesting from a Surf Sci point of view because some of their properties as surface active proteins are quite spectacular. In this review, recent advances in understanding these properties will be surveyed. We will attempt to define what the properties are that make them unique. As an understanding of both structure and function of hydrophobins is emerging we see that this is paving the way for industrial applications as well as an understanding of their biological functions. Major recent advances: Recently there has been a clear increase in attempts to use hydrophobins in applications. We are starting to understand their unique properties as surfactants and especially applications related to the stability and development of foams and various surface treatments are emerging. There are several new reports on molecular structures as well on mechanisms of self-assembly. Hydrophobins have functions in biology that are far from understood, but also here techniques are developing and a broader understanding is emerging. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Hydrophobins are proteins that are produced by filamentous fungi. An example of such a fungus is the common button mushroom, Agaricus bisporus, which is a common part of our normal diet. Hydrophobins can be secreted out in the surroundings or retained in the fungal structures, such as fruiting bodies or mycelium. Their biological functions seem to be diverse, but always seem to relate to interactions with interfaces or surfaces in some manner. We still do not have a good way of predicting if a hydrophobin will be found in the surrounding medium, or if it is associated with the fungal structures. However, it seems that in whichever context a hydrophobin is found, it plays a role as a coating/protective agent, in adhesion, surface modification, or other types of function that require surfactant-like properties. Hydrophobins were originally found when studying highly expressed genes in filamentous fungi, and in their pioneering work, Wessels found proteins associated with the genes. The initial work on hydrophobins has been covered in several excellent reviews (see below) and will not be covered further here. For understanding the current research on hydrophobins it is essential to know that early results led to the classification of hydrophobins into two classes, class I and class II [1]. The initial comparison of properties and sequences showed that the sequences could be classified based on the occurrence of hydrophilic and hydrophobic amino acid residues in

the protein sequence i.e. according to their hydropathy plots [2]. The members of the group named class I shared a functional similarity in that the aggregates that they formed were highly insoluble in aqueous solution, whereas the members of class II formed aggregates that were much easier to dissolve. However, the sequences of both class I and II hydrophobins share a distinguishing feature. In their sequences there are typically eight Cysresidues in a special pattern. These Cys residues are in a sequence such that the second and third Cys residues follow each other immediately in sequence, forming a pair. The sixth and seventh also form a similar pair, but the rest of the Cys residues do not have other Cys residues as near neighbours. This pattern (separated, pair, separated, separated, pair, separated) has a striking symmetry and can be easily recognized in a primary sequence. The different classes of hydrophobins are found in the taxonomic group of fungi so that both class I and II are found in Ascomycetes, while only class I have been found in Basidomycetes. For more detailed sequence comparisons the reader is referred to other reviews [3,4]. In this review the most recent literature is reviewed. There are several excellent previous reviews that explore earlier work and especially if the reader is interested in background information in evolutionary relationships, phylogeny and biological roles, this earlier literature should be consulted [1,3–9].

2. Structure of hydrophobins ⁎ Tel.: +358 20 722 5136; fax: +358 20 722 7071. E-mail address: markus.linder@vtt.fi. 1359-0294/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cocis.2009.04.001

An important step towards understanding how hydrophobins function came with the first crystallographic structure [10••]. This was

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are much larger in EAS, indicating a structural reason for the observed differences in function. The authors also suggest how individual EAS molecules could pack together forming the rodlet structures that are characteristic for EAS and other class I hydrophobins.

3. What are the special properties of hydrophobins? 3.1. Formation of surface membranes

Fig. 1. A) Structure of HFBII. The hydrophobic patch is shown in green, with the rest of the hydrophilic surface in light grey. The exposed hydrophilic side chains give the molecule the character of an amphiphile. B). Two pairs of disulfide bridges span the structure.

of the class II hydrophobin HFBII from Trichoderma reesei.1 This structure, which is shown in Fig. 1, came as a surprise because none of the symmetry of the Cys residues seen in the sequence could be seen in the structure at first. Briefly the structure can be described as being nearly globular, having a central β barrel structure and a small segment of α helix. The diameter of the protein is approximately 3 nm, and its molecular weight is 7.2 kDa. The most notable feature of the protein was that one part of the surface consisted nearly entirely of hydrophobic aliphatic side chains, forming what was called “the hydrophobic patch”. A few things are notable about this patch. When comparing the sequences of HFBII to other class II hydrophobins, it could be noted that the residues forming this patch are conserved, that is, similar residues are found in the corresponding positions in the other sequences. Because they have been conserved in evolution, this indicates an important functional role. The patch is formed to a large extent by two loop regions in the central beta barrel structure. The patch contains only aliphatic residues, not any aromatic ones which also are hydrophobic. The significance of this is still not clear. Also, these surface hydrophobic residues form about half of all hydrophobic residues in the protein. This is remarkable, because usually hydrophobic residues are buried in the core of proteins stabilizing the folded conformation of the protein. In short, the structure could then be described to look like any surfactant with one hydrophobic and one hydrophilic part, only that the size and structural details are very different from typical surfactants. Instead of having a core stabilized by hydrophobic interactions, HFBII has an extended network of disulfide bonds that stabilize the structure and essentially forms the core of the protein (Fig. 1B). The two paired Cys residues are in the middle of the protein, allowing the sort of “double” disulfide bonds to be formed that span the entire structure. The disulfides seem essential for the structure, but remarkably it was shown for the class I hydrophobin SC3 from Schizophyllum commune that functionality was retained even after reduction and blocking of them [11•]. The most detailed structural work on a class I hydrophobin has been obtained for the EAS hydrophobin from Neurospora crassa[12••]. In this work by Kwan et al. the solution structure was studied by NMR. It was shown that EAS and HFBII share the same fold despite a very low sequence similarity. It was concluded that the disulfide bridging pattern was the same as in HFBII. In Fig. 2 a superimposition of the two structures and that of HFBI [13•], also from T. reesei are shown. From this superimposition it can be seen that the part of the protein that in HFBII was described as a hydrophobic patch looks different in EAS. The two loops that form between the strands in the beta barrel structure 1 T. reesei has been identified as an asexual form of the fungus Hypocrea jecorina. Therefore it should be preferred to use the name H. jecorina instead of T. reesei. However, to avoid confusion when comparing to literature, we will use T. reesei in this review.

Hydrophobins have been described as the most surface active proteins known, and they certainly exhibit some unique properties in terms of the nature of surfaces and interfaces which they can assemble to form. There are a number of publications reporting surface tensions of aqueous solutions of hydrophobins. Values reported range from 45 to 27 mNm− 1, depending on the hydrophobin type and concentrations used [14,15]. However, measuring surface tension of hydrophobins has not been very easy. For example, different drop-shape techniques are commonly used, but as was shown for HFBI from T. reesei, the hydrophobin actually changes the shape of the drop so radically that these techniques were simply not applicable [16•]. Cox et al. [17••] used the Wilhelmy plate method to measure the surface tension and obtained reproducible results at low hydrophobin concentrations, obtaining maximal reduction of surface tension to about 25 mN m− 1 for HFBI and HFBII. Plots of concentration versus surface tension showed that there was a clear break in the curve at protein concentrations of 0.38 µM. The authors described this point as the surface saturation concentration (SSC). This term was suggested to describe the behaviour of the hydrophobins better that the critical micelle concentration (CMC) because there appears to be no direct connection between the SSC and the formation of oligomers in solution. Szilvay et al. came to the same conclusion using engineered proteins with dramatically changed solution aggregation behaviour [18]. In this work no change in SSC was observed, although the solution oligomerization was increased by orders of magnitude. However, it seems that the importance of another observation is becoming more evident. As noted above, the hanging-drop method to determine surface tension is not applicable to hydrophobin solutions because of the irregular shape that drops attain, and which seemed to be caused by the formation of a membrane [16•]. Similarly Cox et al. [17••] noted that at higher concentrations the Wilhelmy plate was locked in place by a “skin”, or membrane, that the hydrophobin formed, and which complicated measurements. This membrane formation is most likely connected to another observation which was made in the same work. Measurements of surface shear rheology were performed to quantify surface elasticity and viscosity. It was noted that

Fig. 2. Structural superimposition of EAS (yellow), HFBI (purple) and HFBII (green). All hydrophobins share the same basic fold. Large differences are however found in the loops of the β-hairpins of the central β-barrel (indicated by arrows).

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especially the elasticity was remarkably high, about 0.5 Nm− 1, which is orders of magnitude higher than observed for any other surface active proteins. Surface shear rheology measurements have not been reported for class I members, but it is clear that membranes are also formed by these [19•]. The high surface elasticity of membranes of hydrophobins is connected to their tendency to form very stable foams, another property that seems unique to hydrophobins. The foaming tendency may be stronger for class II members than for class I members, for which systematic studies have not been published. The foaming capability of hydrophobins has been known already for some time, as noted for example by studying phenotypes of deletion strains [20]. Bubble stability of HFBII was studied by Cox et al. [21•] who found that foams and bubbles of HFBII from T. reesei were stable for at least 4 months, and even up to several years in some cases, where the concentration of protein used was relatively low (0.1 wt.%). A discussion on hydrophobins versus other means of stabilizing foams can be found in the review by Murray [22].

3.2. Foam formation One interesting observation that is receiving an increased amount of attention is the relation between gushing of beer and hydrophobins. Beer gushing is a phenomenon where beer foam gushes out of the bottle when it is opened, almost emptying it even if it has not been shaken before. Such products cannot be sold to the consumers and therefore poses a problem for the brewers and malt producers. Sarlin et al. [23•] showed that there is a clear connection between the phenomenon and the presence of hydrophobins. It is remarkable that very low amounts of hydrophobins are sufficient for gushing to occur, amounts in the order of micrograms per 33 cl bottle is enough. One must therefore conclude that the gushing described by Sarlin is not the same thing as the foam stability described by Cox. The gushing foam is not exceptionally stable, but is formed very effectively and instantly. Gushing is therefore more likely to be related to the hydrophobins acting as nucleation sites for the formation of CO2 bubbles. Recent progress has been made by Zapf et al. who cloned hydrophobin genes from Fusarium culmorum, that are widespread in causing gushing problems [24]. Interestingly the found hydrophobins belong to class II, which may indicate that gushing is more related to class II than class I members.

3.3. Rodlet formation The capability to form rodlets is one of the early observations that were made for class I hydrophobins [1]. They have a characteristic appearance in images from electron microscopy and atomic force microscopy (Fig. 3A). Rodlets are typically formed when a solution of hydrophobin is dried down on a solid surface, and appear to be formed at the air–water interface. Rodlets also have interesting analogies with amyloid fibres [25]. Because of their characteristic appearance they have been used to identify hydrophobin assemblies on the surface of fungal structures. This is illustrated by the example of Dague et al. [26] who were able to draw conclusions on the composition of fungal cell surfaces structures based on the observation of rodlets. The rodlets are very difficult to dissolve and typically neat trifluoro acetic acid had been used for the purpose of dissolving the rodlets. Kwan et al. made progress in this field when they used X-ray diffraction and NMR structural data to propose a molecular mechanism for the assembly of the rodlets of the EAS hydrophobin from N. crassa [12••]. The X-ray studies showed a characteristic 4.8 Å reflection that is indicative of βstructure, but were missing the 10–12 Å reflection that are typically found in amyloidal fibres and indicate stacked β-sheets. This indicated that the mechanism of rodlet formation does not like a typical amyloid fiber involve structural rearrangement into stacked β-structures but that the core structure of the EAS would be essentially intact [12••]. Other recent observations of rodlets include Yu et al. [27] who studied the formation of rodlets of the HGFI hydrophobin from Grifola frondosa. They used a Langmuir trough and found that rodlets formed at the air–water interface during compression of a surface film of HGFI. When a membrane of HGFI was first formed on the air–water interface no rodlets could be seen. However, images of the membrane after repeated compression cycles showed densely packed rodlets. This observation is supported by findings by Houmadi et al. [28] who studied a hydrophobin from Pleurotus ostreatus. They propose a mechanism for rodlet formation through a bilayer intermediate based on mathematical analysis of compression data from experiments with a Langmuir trough. These studies show that rodlets are structures that form as a final stage from other intermediate assembled structures. Thus, a picture is emerging that class I and II form similar surface membranes initially, but for class I the films turn into insoluble rodlet films whereas class II hydrophobins can dissociate again reversibly. For a discussion on the function of amyloids in microorganisms, see the review by Gebbink et al. [29].

Fig. 3. A) Atomic force microscopy (AFM) image of rodlets formed by the HGFI hydrophobin form Grifola frondosa. Rodlet formation is characteristic of class I hydrophobins. The rodlets were formed at the air–water interface in a Langmuir trough by multiple compression and lifted on a solid support for imaging as described in [27] B) A surface membrane of HFBI imaged by AFM showing an organized structure. The film was formed at the air–water interface and lifted onto a mica support as described in [45].

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Another conclusion of recent literature is the confirmation of the observation that only hydrophobins of class I produce these insoluble rodlets, as all reports including the ones above deal with class I hydrophobins. For class II hydrophobins various aggregates which have been described as needles or fibrils are formed, but their properties like size, and solubility are different [30,31]. These have not received much attention recently, but were discussed in the recent review by Sunde et al. [25]. The class II aggregates also form in a different way that the class I rodlets. While class I rodlets seem to form when a surface membrane gets compressed or “matures”, the class II aggregates are much larger and found in the solution. For class II aggregates it seems that they could also be originating from the surface membrane, so that they would be pieces of the membrane which have been broken up under shear and rearranged (personal communication with Andrew Cox and observation by the author). 3.4. Surface adhesion The capability of hydrophobins to adhere to various surfaces was one of the early observations for hydrophobin function. An early finding on the behaviour of the class I hydrophobin SC3 was that when it binds to for example Teflon, it can form a very insoluble layer as studied in detail by de Vocht et al. [32•]. It can be especially interesting from a surfactant point-of-view that the membranes are insoluble in sodium dodecyl sulphate (SDS). All hydrophobins adhere to surfaces, but as shown by for example Askolin et al. [14] there is a difference in the binding characteristics. While class I members can be made to adhere very strongly, this is not seen for class II members which dissociate more easily. Although dissociation is easier for class II members, it was shown that the class II HFBI was able to compete efficiently for the binding surface with the class I SC3, resulting in a mixed membrane. 4. How can the structure of hydrophobins explain their properties? Looking at the different applications that are emerging for hydrophobins, we can note that much of them are related to the ability of hydrophobins to form interfacial layers and membranes, either in the form of foams or films on the surface of for example sensors. However, it is still not really known what it is that gives these special properties to hydrophobins (Fig. 4). Understanding the structure–function relations in hydrophobins would give a clearer basis for using them in applications and understanding their function in biology. We can see clearly that different

Fig. 4. Self-assembly of hydrophobins. In an aqueous solution, schematically shown here as a drop on a solid surface, hydrophobins show different types of interactions. In solution, different types of oligomeric assemblies are formed, most likely a mixture of monomers, dimers, and tetramers. At the air–water interface hydrophobins assemble into films that can have a very ordered structure. Hydrophobins also bind to solid surfaces, forming films that can be very tightly bound.

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hydrophobins differ in certain properties, possibly with a gradient of properties between the extremes of class I and class II. A better understanding of structure–function relations would allow understanding their biological roles better and choosing a suitable one for applications as well as perhaps designing new ones with specific desired properties. An overall model for how hydrophobins function has emerged (Fig. 4). Briefly this model is that hydrophobins are soluble in solution by forming different dimers and oligomers. These oligomers disassociate at interfaces and the hydrophobin rearranges to form surface membranes [33–36]. For class I hydrophobins especially, the membrane formation may involve several rearrangements, ending up in the characteristic rodlet structures [37]. Below we will discuss some more recent advances in understanding this process. Looking at the high resolution structures available, one of the most striking features is the hydrophobic patch exposed on one side of the protein. It clearly exhibits some surfactant like characteristics in the sense that the molecule is amphiphilic, but does this completely explain their properties. Here it is interesting to compare hydrophobins to another group of compounds that are being studied. These are the so-called Janus particles which are nano- or micron sized particles that have two surfaces of different polarity. The study of Janus particles has actually drawn inspiration from hydrophobins [38]. In fact, Janus particles also assemble into unexpected aggregates. The energy to remove these particles from an interface is much larger than for smaller molecules and therefore they are more efficient at stabilizing interfaces. The analogy to Janus particles was also discussed by Cox et al. [17••]. It is also noteworthy that it has been demonstrated that the size of a hydrophobic particle has an effect on the solvation free energy per unit area [39]. As the size of a hydrophobic particle grows, this will affect how the water molecules can arrange around this particle. At a certain size the hydrogen bonding network cannot adapt around the hydrophobic particle and the water molecules will move away from the hydrophobic object. This means that the free energy of solvation per unit area increases when the size of the hydrophobic object increases. This scaling effect could contribute to the special properties of hydrophobins compared with traditional small molecular weight surfactants. However, it is well known that the geometry of surfactants affects their behaviour in several ways. The relative size and geometry of the hydrophilic and hydrophobic head groups, i.e. their “packing” properties, affect for example the size and geometry of micelles [40]. Because hydrophobins have a defined size and structure there are possibilities for specific interactions between molecules. These should affect the geometry and structure of aggregates both in solution and on interfaces. Some recent work has brought some understanding on the structure of these assemblies. Szilvay et al. [16•] used atomic force microscopy (AFM) to study membranes of wild-type and engineered variants of HFBI (class II) that had formed on the air–water interface. Membranes were studied both from their hydrophilic side as well as their hydrophobic sides, by using different techniques to deposit the membranes on solid supports. A very regular structure could be seen with details down to the nanometer range (Fig. 3B). The thickness of the layer corresponded to that of a single protein molecule. However, individual molecules could not be identified. X-ray diffraction work on similar membranes confirmed that the packing was hexagonal with a lattice constant of 5.4 nm [41,42]. The data therefore point to the direction that the membranes formed by hydrophobins have a very regular crystalline structure [43•]. This indicates specific cohesive interactions between the molecules, and these interactions may very well be a contributing reason for the high viscosity of the membranes. Wang et al. studied the membrane formed by SC3 from S. commune and found that the membrane it forms was permeable to water but not to molecules with a MW over 200 g mol− 1 [44]. This observation is in accordance with a structurally defined membrane.

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Hydrophobins can be very soluble in water, and for example X-ray diffraction experiments have been performed with samples with concentrations up to 100 mg/ml [45]. The formation of different self assembled macroscopic assemblies was discussed earlier, but there are much data that show that also in clear solutions the hydrophobins are present in some self-assembled structures [35,36]. Kisko et al. [46] used small angle X-ray scattering to study the formation of oligomers in solution by HFBI and HFBII. By changing solution conditions it was found that hydrophobic interactions play an important role in the formation of these assemblies. While there are data suggesting lateral interactions between hydrophobins at the air–water interface, there are still not much data that describe the molecular details of how hydrophobins interact in solution. One interesting observation, however, comes from high resolution X-ray studies of crystallized protein. It is surprising in how many different crystalline packing arrangements have been observed for HFBI and HFBII from T. reesei [10••,13•,34•,47]. While these do not necessarily follow the interactions seen between hydrophobins in solution, we can at least see that there are several different ways in which favourable contacts between the protein molecules are found (Fig. 5). Structural characterization has recently given some new insight in the function of the hydrophobic patch. The crystal structures of two class II hydrophobins HFBI and HFBII from T. reesei have been determined [10••,13•], and NMR has been used to study the structure of EAS from N. crassa belonging to class I [12• •]. As noted above, these structures share a common fold, and HFBI and HFBII are remarkably similar. However, it was surprising that two forms of HFBI could be seen. These forms differed in the loops that comprise the hydrophobic patch. One form was similar to that previously seen in HFBII, but in the other the loops were more extended. The structural analysis of EAS revealed that its corresponding loops were still much more extended. There is thus a clear plasticity in these loops, and it is highly likely that this plasticity can be very important for the function of hydrophobins. Molecular modelling of SC3 (class I) from S. commune also suggested the importance of particularly the first beta hairpin loop (corresponding to the segment between the third and fourth Cys residue) in binding to interfaces, in an analogous way as had been suggested for HFBII [33]. The studies on EAS were continued by making changes in these loops to get a better understanding of how they function [48••]. In this work the loops were truncated in several mutants, deleting up to 17 residues of the 25 residue loop. It was surprising to note that the deletion mutant was still capable of

Fig. 5. Hydrophobins have shown several different packing arrangements in crystals used for structure determination [34•]. In this example for HFBII, the protein has formed a sheet-like layer with the hydrophobic patch of each molecule facing the same way. Individual molecules pack towards each other by lateral interactions forming a twodimensional network. Although the molecular details are not known, similar packing could also occur at interfaces, and explain such behaviour as viscoelastic properties and the stability of surface bound films. Ordered packing are also seen by AFM in surface films as shown in Fig. 3B and by diffraction techniques [42].

forming rodlet structures showing the same insolubility as the wild type. Deletion mutants were also capable of forming coatings on Teflon with the same wettability properties as the wild type. It is very interesting that hairpin loops are flexible in examples of both class I and class II hydrophobins, but it seems that a large variation in length can be accommodated. The dynamics of interaction in solution for HFBI was studied by Szilvay et al. The dependence of solution concentration for the oligomerization state was demonstrated using fluorescence resonance energy transfer [35], and it was shown that oligomerization increases with increasing protein concentrations. However, in a subsequent work the surprising finding was made that the equilibrium of oligomerization does not affect the capability of the protein to reduce surface tension. Protein variants that had been linked together by peptide linkers had an increased oligomerization capability, but nearly identical reduction of surface tension [18]. This could indicate that interfacial adsorption does not occur via monomers, but that oligomers could directly adsorb at the interfaces. A comparison of the surface activity of hydrophobins to other proteins which are used for their surface activity should also be made. One can say that most proteins are more or less surface active. For example, when whey proteins are heated they de–nature and expose hydrophobic parts and become surface active. To clarify the above discussion, the view of how hydrophobins function is that surface activity is a property of the folded protein. In fact, hydrophobins are very hard to denature, as shown for example by Askolin et al. [14] who heated hydrophobins to 90 °C without any sign of denaturing. There may be some change in shape for hydrophobins when they selfassemble, but this is a part of their function, and it should not be confused with denaturing. 5. Applications of hydrophobins One of the recent trends that we see with hydrophobins is their progress towards industrial applications. This can be seen by the large number of patent applications that have been filed recently. In this review the patent applications will not be discussed. However, published summaries such as that by Subkowski et al. [49] show that a wide variety of potential uses of hydrophobins in applications such as the formation of coatings and removal of diesel and oil from contaminated water. Examples are also found for various dispersion applications and for stabilizing emulsions [15]. Large efforts are also made to produce hydrophobins in industrial scale [50]. Here only the most recent publications are examined but more examples are found in earlier reviews [3,6]. A different, but clearly expressed and interesting application is to use hydrophobins in the manufacture of aerated foods such as ice cream [51]. Ice cream is described as a “…multiphase structure consisting of ice, air, and fat…”. In this application the role of the hydrophobin is to stabilize the dispersed air bubbles. This use is analogous to the use of an ice structuring protein that can be used to control the structure of the ice component [52]. There is a clear interest in materials technology to control the structures of materials at the molecular level and find new techniques in the manufacture of high-performance materials. Here the attention is often turned to biology for both inspiration and components. In the work by De Stefano et al. [53] it was found that the surface films formed by a class I hydrophobin from Pleurotus ostreatus were stable in KOH, and thus they could demonstrate the use of this hydrophobin as a new material in a silicon micromachining process. When patterns of this hydrophobin were placed on silicon, they bound to the silicon and protected it from etching with KOH. Thus non-covered parts were etched and patterns formed. The same group continued to study interactions with porous silicon and found that the wettability of the porous silicon could be modified by the hydrophobin and propose the use of this method to passivate optical devices [54].

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The surface adhesion and surfactant properties of HFBI were used by Kurppa et al. [55] who found that it could efficiently dissolve single walled carbon nanotubes in water. They continued by using HFBI labelled with small (1.4 nm) gold nanoparticles to show that the binding of HFBI to the nanotubes could actually be seen by the use of electron microscopy as the ordered arrangement of the gold particles along the nanotubes. The spacing and arrangement of the nanoparticles were in accordance with the dimensions of HFBI. The surface adhesion of HFBI was also utilized by Kostiainen et al. [56] who made conjugates of cationic dendrons with a variant of HFBI that was engineered to contain a single additional sulfhydryl in an added N-terminal Cys-residue. Dendrons are highly branched macromolecules that are made by an exact synthesis route. In this case the designed property of the dendron was to bind DNA, and it was shown that the molecular hybrid had the hydrophobins' adhesion properties and the dendrons DNA binding property. This combination had a surprisingly high efficiency in DNA transfection experiments [57]. Misra et al. [58], have identified that some personal care and biomedical applications such as catheters require polymeric materials with surfaces that have low friction. They coated polymers such as polystyrene with the class I hydrophobin SC3 from S. commune using spin coating or direct deposition. The deposited surfaces showed a bumpy structure, but this structure was clearly different from the characteristic rodlet structure. They demonstrated that the coated surfaces had a 70–80% reduction in friction coefficient compared to the untreated polymers surface. The application of hydrophobins for surface modification and as surface membranes in biosensing and electrochemical applications is a very active and promising field. Here hydrophobins of both classes have been used successfully. Corvis et al. [59] showed that SC3 from S. commune has a strong interaction with lipid layers which was suitable for surface immobilization. The properties of this layer were demonstrated using SC3 which had been oxidized by performic acid and which could bind CuII-ions. The redox properties of these immobilized ions were then investigated. This work was then extended to show that fullerene derivates also could be incorporated in a SC3 matrix, which allowed efficient electron transfer from the electrode to the fullerene. Previous work on the use of the class II hydrophobin, HFBI from T. reesei for immobilization [60] has found new uses as shown by Zhao et al. [61]. HFBI was simply adsorbed onto polished platinum electrodes, thereafter this surface was used to adsorb glucose oxidase. It was shown that using the HFBI treated surface, glucose oxidase stayed active for longer times on the surface as compared to the bare electrode. Similarly, chicken IgG could be immobilized on HFBI [62] by non-specific adsorption. 6. Biological function of hydrophobins To understand the biological role of hydrophobins remains a challenge even after a large effort by several groups for several years. Roles in protection, adhesion and growth have been shown in earlier work [7] and it seems clear that we will not find single defined roles for hydrophobins, but it seems that they are a family of proteins that have evolved to manage a variety of interfacial interactions and properties for the fungi. Fungi play a huge role in the carbon cycle and have a crucial role in ecology. Fungi are also industrially and economically important. We use them as biocontrol agents, for production of foods and enzymes, and for example for breaking down cellulose for biofuels. They are also harmful, as pests and disease causing agents, they can destroy buildings etc. Many of these roles involve how they interact with their environment and here some hydrophobins may play a key role. An interesting suggestion was made by Rillig [63•] that hydrophobins secreted by fungi in to the soil might actually cause the soil to become hydrophobic and water repellent. This was based on the

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observation that hydrophobins are resistant to degradation and that there are several previous reports that hydrophobins can turn hydrophilic surfaces into hydrophobic ones. It seems logical that this effect on soil would be more affected by class I hydrophobins since their aggregates are more recalcitrant than the class II aggregates. However it seems that real experimental evidence for the theory is still lacking [64]. One new function for the binding of hydrophobins to solid surfaces was proposed by Takahashi et al. [65••]. They studied how Aspergillus orysae degraded a polymer, polybutylene succinate-coadipate (PBSA). This is of interest because PBSA is a much used industrial biodegradable plastic. The enzyme mainly responsible for the hydrolysis of the PBSA was a cutinase, CutL1. However, it was noted that a hydrophobin RoIA was also induced. Further studies showed that the hydrophobin bound to the polymer and actually caused more cutinase to bind, thereby increasing the overall hydrolysis efficiency. This was described as a recruiting function for hydrophobins. This is a very interesting hypothesis, again pointing towards an all-around role for hydrophobins in controlling events at interfaces. Several functions for hydrophobins in the life cycle of fungi have been described and suggested. These include formation of protective layers, attachment, structural components of cell walls, and lowering surface tension to allow aerial growth. One observation that is linked to the multiple roles of hydrophobins is that several different hydrophobin coding genes are typically found in the genomes of fungi. Recent work has brought more understanding of these functions and new methods have been introduced for more systematic studies. In one study, expressed sequence tags were used to study gene expression during different developmental stages in the edible oyster mushroom, Pleurotus ostreatus [66]. In Beauveria bassiana, hydrophobins were pointed out to have key roles in adhesion and for cell surface properties [67]. The role of a class II hydrophobin, VDH1, from the wilt fungus Verticillium dahliae was studied by Klimes and Dobinson [68]. It was found that VDH1 was important for a morphogenic process, the formation on structures called microsclerotia, which are important for the persistence of the fungus in the soil. VDH1 was not needed for the disease development itself, but affected in a more general way the long term survival of the pathogen. In another study, Kershaw et al. [69•] made changes to the Cys-residues in the MGP1 hydrophobin from Magnaporthe grisea. It was found that the correct formation of disulfides was not essential for the production and self-assembly of MPG1 in all cases, but the mutations had a severe effect on aerial growth and sporulation. The presence of hydrophobins in the extracellular matrix of fungi has been demonstrated in several studies, for example in Aspergillus fumigatus which cases respiratory problems in people [70]. It is typical that fungi have genes for several different hydrophobins, for example the tomato pathogen Cladosporium fulvum has six genes for hydrophobins. Lacroix et al. [71•] used a technique where a tag was genetically fused to each of them in turn. This allowed the study of the different roles of each of them. It was found that they localized differently to different parts of the fungus, and that one of them (HCf-6), was observed during infection and had a direct role in adhesion. The presence of hydrophobins in fungal structures has also been measured using mass spectroscopy (MS). It has been found that the hydrophobins are easily detected by MS and that it is convenient to use reduction of the disulfides as a means of identifying them. The difference in mass between the intact and reduced forms should be eight, corresponding to the presence of eight Cys residues involved in disulfides. Using this technique Neuhof et al. [72] showed that the main peaks that were obtained when whole cells were subjected to MS were hydrophobins. This opens new possibilities to study expression and post translational processing of hydrophobins. Additionally because of the ubiquitous presence of hydrophobins, the intact cell MS technique can be used as a fingerprint to identify various strains as discussed further by Degenkolb et al. [73].

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7. Future perspectives In the view of the intensive current industrial interest in hydrophobins, it is likely that hydrophobin based products will be on the market fairly soon. Hydrophobins are an example of a biomolecule that outperform synthetic molecules in many properties. Because they are bio-based, use of hydrophobins also supports a sustainable biotechnology based production. In addition to learning how to utilize the properties of hydrophobins in the most efficient way, it is probably the development of production technologies that will be critical for commercial success of hydrophobin based technologies. One way is to use recombinant bacteria or yeast. However, filamentous fungi are already used extensively as production hosts for industrial proteins. Production levels of tens of grams per litre are for example reported for cellulose degrading enzymes. However, because of the special properties of hydrophobins it is probably not a simple task to increase production levels, because of increased foaming etc. Probably a feasible production still requires a better understanding of how hydrophobins function and how to control their interactions. The research on hydrophobins is at a stage where real progress in understanding structure function relations is possible with two main aspects to be further addressed as follows. What the molecular mechanisms behind the extraordinary properties? Can we explain the surface activity and aggregation properties of hydrophobins in terms of Janus particles or is the mechanism more complicated? When efficient production methods are available it is very likely that we will see hydrophobins as high performance components in a number of applications. Molecular engineering with fusion proteins can give endless variations of functionality. This development is well in line with the current trend of producing smarter chemicals in an environmentally sustainable way. Hydrophobins are a good example of emerging products in industrial biotechnology [74]. We may also see that hydrophobins will inspire the writing of new chapters in textbooks of surface and colloid chemistry. Acknowledgements The work was supported by the Academy of Finland (grants #113436, # 118519). Andrew Cox, Arja Paananen and Geza Szilvay are thanked for useful discussions and comments on the manuscript. Arja Paananen, Johanna Kallio, and Geza Szilvay are thanked for help with the figures. References [1] Wessels JGH. Developmental regulation of fungal cell-wall formation. Annu Rev Phytopathol 1994;32:413–37. [2] Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982;157:105–32. [3] Linder MB, Szilvay GR, Nakari-Setala T, Penttila ME. Hydrophobins: the proteinamphiphiles of filamentous fungi. FEMS Microbiol Rev 2005;29:877–96. [4] Whiteford JR, Spanu PD. Hydrophobins and the interactions between fungi and plants. Mol Plant Pathol 2002;3:391–400. [5] Elliot MA, Talbot NJ. Building filaments in the air: aerial morphogenesis in bacteria and fungi. Curr Opin Microbiol 2004;7:594–601. [6] Hektor HJ, Scholtmeijer K. Hydrophobins: proteins with potential. Curr Opin Biotechnol 2005;16:434–9. [7] Kershaw MJ, Talbot NJ. Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis. Fungal Genet Biol 1998;23:18–33. [8] Scholtmeijer K, Wessels JGH, Woster HAB. Fungal hydrophobins in medical and technical applications. Appl Microbiol Biotechnol 2001;56:1–8. [9] Wosten HAB, de Vocht ML. Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta, Bioenerg 2000;1469:79–86. [10] Hakanpaa J, Paananen A, Askolin S, Nakari-Setala T, Parkkinen T, Penttila M, et al. •• Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. J Biol Chem 2004;279:534–9. (The molecular structure of a hydrophobin is for the first time described). [11] de Vocht ML, Reviakine I, Wosten HAB, Brisson A, Wessels JGH, Robillard GT. • Structural and functional role of the disulfide bridges in the hydrophobin SC3. J Biol Chem 2000;275:28428–32. (Surprising findings on the effect of modifying disulfide bridges are described).

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