Markus B. Linder

The roles and function of cellulose-binding domains

Most cellulolytic enzymes consist of distinct catalytic and cellulose-binding domains (CBDs). Similar domain structures are also found in enzymes degrading other insoluble carbohydrates such as raw starch and chitin. Such binding domains improve the binding and facilitate the activity of the catalytic domain on the insoluble but not on soluble substrates. Based on their amino acid sequence similarities, the CBDs have been divided into several different families. Structure determination and subsequent mutagenesis studies have revealed that CBDs rely on several aromatic amino acids for binding to the cellulose surfaces. The CBDs binding to crystalline cellulose have different topologies but share similar rigid backbone structures for correct positioning of the side chains required for the substrate recognition and binding. CBDs represent ideal affinity tags for specific immobilisation of various other proteins to cellulose. Furthermore, improved understanding and control of their action will be important for the improvement of the biotechnological value of cellulolytic enzymes.

The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules.

Cellulose binding modules (CBMs) potentiate the action of cellulolytic enzymes on insoluble substrates. Numerous studies have established that three aromatic residues on a CBM surface are needed for binding onto cellulose crystals and that tryptophans contribute to higher binding affinity than tyrosines. However, studies addressing the nature of CBM–cellulose interactions have so far failed to establish the binding site on cellulose crystals targeted by CBMs. In this study, the binding sites of CBMs on Valonia cellulose crystals have been visualized by transmission electron microscopy. Fusion of the CBMs with a modified staphylococcal protein A (ZZ-domain) allowed direct immuno-gold labeling at close proximity of the actual CBM binding site. The transmission electron microscopy images provide unequivocal evidence that the fungal family 1 CBMs as well as the family 3 CBM from Clostridium thermocellum CipA have defined binding sites on two opposite corners of Valonia cellulose crystals. In most samples these corners are worn to display significant area of the hydrophobic (110) plane, which thus constitutes the binding site for these CBMs.

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 Å resolution. HFBII has a novel, compact single domain structure containing one α-helix and four antiparallel β-strands that completely envelop two disulfide bridges. The protein surface is mainly hydrophilic, but two β-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 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.

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.

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.

Interfacial Engineering by Proteins: Exfoliation and Functionalization of Graphene by Hydrophobins

Graphene has attracted vast interest as a new material with many uses. 2] Two-dimensional, crystalline graphene has many advantageous properties, such as extremely high electric and thermal conductivity, high strength, and a large surface area. Many more useful properties can result from graphene assemblies and modification by different functionalities or additional molecules. One of the usual ways to functionalize graphene is chemical modification; however, attempts to modify the surface of graphene in a noncovalent, nondestructive way have also been successful. These methods typically involve the buildup of charge on the graphene surface to enable the stabilization and assembly of the graphene sheets on the basis of electrostatic interactions. In a further step towards more complex functionalities, we have now modified graphene with more specifically interacting coatings consisting of biomolecules. One of the main challenges in the production of graphene is the scalable, controllable, and safe processing and handling of individual graphene sheets. Methods for the fabrication of graphene in a dry environment include the micromechanical cleavage of graphene sheets from graphite and the epitaxial growth of graphene on certain substrates. 11] By these methods, very large entities of single-layer graphene can be produced, but the scalability and handling problems remain. High-yielding solution-based chemical methods that enable the handling of graphene in dispersed form have been proposed; however, they involve the direct oxidation of graphene, which may lower the conductivity of graphene dramatically. Recent reports on the exfoliation of graphene either in pure solvents or in the presence of surfactants offer promise for the production of graphene. The main benefits of solution methods are the better processability and increased safety of graphene when it is dispersed in a liquid instead of being used as a dry powder. The dispersion of graphene into aqueous solutions is especially attractive because of their nonvolatile nature. Herein, we present a method for the exfoliation and functionalization of graphene sheets by an amphiphilic protein. It is known that a microbial adhesion protein, HFBI (Figure 1a), which belongs to a class of proteins called hydrophobins, interacts strongly with hydrophobic surfaces, such as graphite and silicon. The protein has a strongly cross-linked fold containing four disulfide bridges. Its most striking feature is a patch of hydrophobic residues on one face of its structure. Thus, the protein resembles a typical surfactant with a hydrophilic and a hydrophobic part. In solution, hydrophobic interactions between individual proteins lead to the formation of dimers or tetramers. In the vicinity of the interface between water and air, however, assembly of the protein at the interface is strongly preferred, and the protein crystallizes as a 2D lattice. Lateral interactions between surface proteins at interfaces may lead

Genetic Engineering of Biomimetic Nanocomposites: Diblock Proteins, Graphene, and Nanofibrillated Cellulose

Nature has materials with extraordinary stiffness, strength, and toughness that is based on aligned, tailored self-assemblies. They have inspired biomimetic nanocomposites with drastically better properties than synthetic composites. Herein we show a new approach to making biomimetic nanocomposites based on the exfoliation of graphite into a matrix of genetically engineered proteins and native nanofibrillated cellulose. The protein was genetically engineered to incorporate a hydrophobin block, which binds to graphene, and a cellulose-binding block, which binds to nanofibrillated cellulose, thereby bringing about both the self-assembly and adhesion between the nanoscale components. The aligned co-assembly leads to remarkably good mechanical properties (modulus: 20.2 GPa, strength: 278 MPa, strain-to-failure: 3.1 %, and work-of-fracture 57.9 kJ m ). The bifunctional protein was crucial for the excellent mechanical properties. This concept shows how high-performance biomimetic composites can be built through the binding and self-assembly of advanced biomolecules that have been genetically tailored. Biology shows numerous composite materials wherein aligned hard and soft self-assembled components are bound together to result in excellent mechanical properties such as the combination of toughness, strength, and stiffness. Such materials are, for example, nacre, plant tissue, bone, silk, and tendon. Factors contributing to their advantageous properties include the chemical nature of the hard-reinforcing and soft-dissipating components, their molecular interactions, their mechanical interlocking, dimensions, and alignment, which contributes to the mechanics of crack propagation. The soft matrix is especially interesting as it acts as glue that keeps the hard components together and allows dissipation of fracture energy. Still, very little is known about, for example, how the matrix proteins of nacre function. A rational route towards a controlled interconnectivity between the self-assembled domains in biomimetic composites is suggested by the design principles of block copolymers, which are used in materials science, for example, to interface two different polymers in mixtures or to stabilize colloidal systems, even for responses or functions. In this work we show the feasibility of genetically engineered proteins having two well-defined binding blocks, denoted as diblock proteins, that bind and assemble the structural components for biomimetic composites. Previously we have shown that the adhesive surfactantlike proteins, hydrophobins, allow exfoliation of graphite to give singleor few-layer flakes of graphene in aqueous solutions. Here, the same route to disperse singleor fewlayer flakes of graphene using proteins in a cellulose matrix was employed to form biomimetic nanocomposite materials. The dispersions of the singleor few-layer flakes of graphene are referred to herein simply as graphene dispersions, although there may be a range of flake thicknesses present. A genetically modified hydrophobin was used to combine graphene and native nanofibrillated cellulose (NFC), also called nanocellulose or microfibrillated cellulose. The structure of the resulting composite resembles that of nacre where self-assembled, aligned platelet-like aragonite reinforcements are embedded in a protein matrix containing nanofibrillar chitin. By using engineered molecules that contain unusual combinations of binding abilities, it is possible to build composites from components that do not occur in natural materials. This technique allowed us to combine flakes of graphene, one of the strongest materials presently known, and nanofibrillated cellulose having a modulus approaching the one of steel 16] in a nanocomposite material. The protein was genetically engineered to connect graphene and NFC, so that it self-assembles at the interfaces, thus leading to cohesion and alignment (Figure 1 a). Binding to graphene was achieved by a hydrophobin, more specifically the class II hydrophobin HFBI, which self-assembles on various interfaces and surfaces, including graphene. Binding to cellulose was achieved by using a protein denoted as a cellulose-binding domain (CBD) found in cellulose[*] Dr. P. Laaksonen, J.-M. Malho, Prof. M. B. Linder Nanobiomaterials, VTT Technical Research Centre of Finland P.O. Box 1000, 02044 VTT (Finland) E-mail: paivi.laaksonen@vtt.fi Homepage: http://www.vtt.fi/research/technology/nanobiotechnology.jsp

The difference in affinity between two fungal cellulose-binding domains is dominated by a single amino acid substitution

Cellulose‐binding domains (CBDs) form distinct functional units of most cellulolytic enzymes. We have compared the cellulose‐binding affinities of the CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from the fungus Trichoderma reesei. The CBD of EGI had significantly higher affinity than that of CBHI. Four variants of the CBHI CBD were made in order to identify the residues responsible for the increased affinity in EGI. Most of the difference could be ascribed to a replacement of a tyrosine by a tryptophan on the flat cellulose‐binding face.

Drug release from nanoparticles embedded in four different nanofibrillar cellulose aerogels

Highly porous nanocellulose aerogels prepared by freeze-drying from various nanofibrillar cellulose (NFC) hydrogels are introduced as nanoparticle reservoirs for oral drug delivery systems. Here we show that beclomethasone dipropionate (BDP) nanoparticles coated with amphiphilic hydrophobin proteins can be well integrated into the NFC aerogels. NFCs from four different origins are introduced and compared to microcrystalline cellulose (MCC). The nanocellulose aerogel scaffolds made from red pepper (RC) and MCC release the drug immediately, while bacterial cellulose (BC), quince seed (QC) and TEMPO-oxidized birch cellulose-based (TC) aerogels show sustained drug release. Since the release of the drug is controlled by the structure and interactions between the nanoparticles and the cellulose matrix, modulation of the matrix formers enable a control of the drug release rate. These nanocomposite structures can be very useful in many pharmaceutical nanoparticle applications and open up new possibilities as carriers for controlled drug delivery.