The complex structure of Fomes fomentarius represents an architectural design for high-performance ultralightweight materials.

High strength, hardness, and fracture toughness are mechanical properties that are not commonly associated with the fleshy body of a fungus. Here, we show with detailed structural, chemical, and mechanical characterization that Fomes fomentarius is an exception, and its architectural design is a source of inspiration for an emerging class of ultralightweight high-performance materials. Our findings reveal that F. fomentarius is a functionally graded material with three distinct layers that undergo multiscale hierarchical self-assembly. Mycelium is the primary component in all layers. However, in each layer, mycelium exhibits a very distinct microstructure with unique preferential orientation, aspect ratio, density, and branch length. We also show that an extracellular matrix acts as a reinforcing adhesive that differs in each layer in terms of quantity, polymeric content, and interconnectivity. These findings demonstrate how the synergistic interplay of the aforementioned features results in distinct mechanical properties for each layer.

Quantifying biomolecular hydrophobicity: Single molecule force spectroscopy of class II hydrophobins.

Hydrophobins are surface-active proteins produced by filamentous fungi. The amphiphilic structure of hydrophobins is very compact, containing a distinct hydrophobic patch on one side of the molecule, locked by four intramolecular disulfide bridges. Hydrophobins form dimers and multimers in solution to shield these hydrophobic patches from water exposure. Multimer formation in solution is dynamic, and hydrophobin monomers can be exchanged between multimers. Unlike class I hydrophobins, class II hydrophobins assemble into highly ordered films at the air-water interface. In order to increase our understanding of the strength and nature of the interaction between hydrophobins, we used atomic force microscopy for single molecule force spectroscopy to explore the molecular interaction forces between class II hydrophobins from Trichoderma reesei under different environmental conditions. A genetically engineered hydrophobin variant, NCys-HFBI, enabled covalent attachment of proteins to the apex of the atomic force microscopy cantilever tip and sample surfaces in controlled orientation with sufficient freedom of movement to measure molecular forces between hydrophobic patches. The measured rupture force between two assembled hydrophobins was ∼31 pN, at a loading rate of 500 pN/s. The results indicated stronger interaction between hydrophobins and hydrophobic surfaces than between two assembling hydrophobin molecules. Furthermore, this interaction was stable under different environmental conditions, which demonstrates the dominance of hydrophobicity in hydrophobin-hydrophobin interactions. This is the first time that interaction forces between hydrophobin molecules, and also between naturally occurring hydrophobic surfaces, have been measured directly at a single-molecule level.

Cellular agriculture - industrial biotechnology for food and materials.

Fundamental changes of agriculture and food production are inevitable. Providing food for an increasing population will be a great challenge that coincides with the pressure to reduce negative environmental impacts of conventional agriculture. Biotechnological manufacturing of acellular products for food and materials has already been piloted but the full profit of cellular agriculture is just beginning to emerge. Cultured meat is a promising technology for animal-based proteins but still needs further development. The concept of plant cells as food offers a very attractive alternative to obtain healthy, protein-rich and nutritionally balanced food raw material. Moreover, cultured microbes can be processed into a wide range of biosynthetic materials. A better control over structural properties will be increasingly important in all cultured cell applications.

Single-Molecule Force Spectroscopy Reveals Self-Assembly Enhanced Surface Binding of Hydrophobins.

Hydrophobins have raised lots of interest as powerful surface adhesives. However, it remains largely unexplored how their strong and versatile surface adhesion is linked to their unique amphiphilic structural features. Here, we develop an AFM-based single-molecule force spectroscopy assay to quantitatively measure the binding strength of hydrophobin to various types of surfaces both in isolation and in preformed protein films. We find that individual class II hydrophobins (HFBI) bind strongly to hydrophobic surfaces but weakly to hydrophilic ones. After self-assembly into protein films, they show much stronger binding strength to both surfaces due to the cooperativity of different interactions at nanoscale. Such self-assembly enhanced surface binding may serve as a general design principle for synthetic bioactive adhesives.

Catch and Release: Engineered Allosterically Regulated ß-Roll Peptides Enable On/Off Biomolecular Recognition.

Alternative scaffolds for biomolecular recognition are being developed to overcome some of the limitations associated with immunoglobulin domains. The repeat-in-toxin (RTX) domain is a repeat protein sequence that reversibly adopts the ß-roll secondary structure motif specifically upon calcium binding. This conformational change was exploited for controlled biomolecular recognition. Using ribosome display, an RTX peptide library was selected to identify binders to a model protein, lysozyme, exclusively in the folded state of the peptide. Several mutants were identified with low micromolar dissociation constants. After concatenation of the mutants, a 500-fold increase in the overall affinity for lysozyme was achieved leading to a peptide with an apparent dissociation constant of 65 nM. This mutant was immobilized for affinity chromatography experiments, and the on/off nature of the molecular recognition was demonstrated as the target is captured from a mixture in the presence of calcium and is released in the absence of calcium as the RTX peptides lose their ß-roll structure. This work presents the design of a new stimulus-responsive scaffold that can be used for environmentally responsive specific molecular recognition and self-assembly.

Observation of pH-Induced Protein Reorientation at the Water Surface.

Hydrophobins are surface-active proteins that form a hydrophobic, water-repelling film around aerial fungal structures. They have a compact, particle-like structure, in which hydrophilic and hydrophobic regions are spatially separated. This surface property renders them amphiphilic and is reminiscent of synthetic Janus particles. Here we report surface-specific chiral and nonchiral vibrational sum-frequency generation spectroscopy (VSFG) measurements of hydrophobins adsorbed to their natural place of action, the air-water interface. We observe that hydrophobin molecules undergo a reversible change in orientation (tilt) at the interface when the pH is varied. We explain this local orientation toggle from the modification of the interprotein interactions and the interaction of hydrophobin with the water solvent, following the pH-induced change of the charge state of particular amino acids.

Hydrophobin-nanofibrillated cellulose stabilized emulsions for encapsulation and release of BCS class II drugs.

The purpose of this study was to construct biopolymer-based oil-in-water emulsion formulations for encapsulation and release of poorly water soluble model compounds naproxen and ibuprofen. Class II hydrophobin protein HFBII from Trichoderma reesei was used as a surfactant to stabilize the oil/water interfaces of the emulsion droplets in the continuous aqueous phase. Nanofibrillated cellulose (NFC) was used as a viscosity modifier to further stabilize the emulsions and encapsulate protein coated oil droplets in NFC fiber network. The potential of both native and oxidized NFC were studied for this purpose. Various emulsion formulations were prepared and the abilities of different formulations to control the drug release rate of naproxen and ibuprofen, used as model compounds, were evaluated. The optimal formulation for sustained drug release consisted of 0.01% of drug, 0.1% HFBII, 0.15% oxidized NFC, 10% soybean oil and 90% water phase. By comparison, the use of native NFC in combination with HFBII resulted in an immediate drug release for both of the compounds. The results indicate that these NFC originated biopolymers are suitable for pharmaceutical emulsion formulations. The native and oxidized NFC grades can be used as emulsion stabilizers in sustained and immediate drug release applications. Furthermore, stabilization of the emulsions was achieved with low concentrations of both HFBII and NFC, which may be an advantage when compared to surfactant concentrations of conventional excipients traditionally used in pharmaceutical emulsion formulations.

Single-Molecule Force Spectroscopy Study on Modular Resilin Fusion Protein.

The adhesive and mechanical properties of a modular fusion protein consisting of two different types of binding units linked together via a flexible resilin-like-polypeptide domain are quantified. The adhesive domains have been constructed from fungal cellulose-binding modules (CBMs) and an amphiphilic hydrophobin HFBI. This study is carried out by single-molecule force spectroscopy, which enables stretching of single molecules. The fusion proteins are designed to self-assemble on the cellulose surface, leading into the submonolayer of proteins having the HFBI pointing away from the surface. A hydrophobic atomic force microscopy (AFM) tip can be employed for contacting and lifting the single fusion protein from the HFBI-functionalized terminus by the hydrophobic interaction between the tip surface and the hydrophobic patch of the HFBI. The work of rupture, contour length at rupture and the adhesion forces of the amphiphilic end domains are evaluated under aqueous environment at different pHs.

Pure Protein Bilayers and Vesicles from Native Fungal Hydrophobins.

Pure protein bilayers and vesicles are formed using the native, fungal hydrophobin HFBI. Bilayers with hydrophobic (red) and hydrophilic (blue) core are produced and, depending on the type of core, vesicles in water, oily media, and even in air can be created using microfluidic jetting. Vesicles in water are even able to incorporate functional gramicidin A pores.

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 ß-sheet structures and assign them to the central ß-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 ß-sheet-specific signals that supports a surface-driven self-assembly mechanism in which the central ß-barrel remains intact and stacks into a larger-scale architecture, amyloid-like rodlets.

The Effect of Hydrophobin Protein on Conductive Properties of Carbon Nanotube Field-Effect Transistors: First Study on Sensing Mechanism.

Hydrophobin is a surface active protein having both hydrophobic and hydrophilic functional domains which has previously been used for functionalization and solubilization of graphene and carbon nanotubes. In this work, field-effect transistors based on single nanotubes have been employed for electronic detection of hydrophobin protein in phosphate buffer solution. Individual nanotubes, single- and multiwalled, are characterized by atomic force microscopy after being immersed in protein solution, showing a relatively dense coverage with hydrophobin. We have studied aspects such as nanotube length (0.3-1.2 µm) and the hysteresis effect in the gate voltage dependent conduction. When measured in ambient condition after the exposure to hydrophobin, the resistance increase has a strong dependence on the nanotube length, which we ascribe to mobility degradation and localization effects. The change could be exceptionally large when measured in-situ in solution and at suitable gate voltage conditions, which is shown to relate to the different mechanism behind the hysteresis effect.

Modular architecture of protein binding units for designing properties of cellulose nanomaterials.

Molecular biomimetic models suggest that proteins in the soft matrix of nanocomposites have a multimodular architecture. Engineered proteins were used together with nanofibrillated cellulose (NFC) to show how this type of architecture leads to function. The proteins consist of two cellulose-binding modules (CBM) separated by 12-, 24-, or 48-mer linkers. Engineering the linkers has a considerable effects on the interaction between protein and NFC in both wet colloidal state and a dry film. The protein optionally incorporates a multimerizing hydrophobin (HFB) domain connected by another linker. The modular structure explains effects in the hydrated gel state, as well as the deformation of composite materials through stress distribution and crosslinking. Based on this work, strategies can be suggested for tuning the mechanical properties of materials through the coupling of protein modules and their interlinking architectures.

Engineering of the function of diamond-like carbon binding peptides through structural design.

The use of phage display to select material-specific peptides provides a general route towards modification and functionalization of surfaces and interfaces. However, a rational structural engineering of the peptides for optimal affinity is typically not feasible because of insufficient structure-function understanding. Here, we investigate the influence of multivalency of diamond-like carbon (DLC) binding peptides on binding characteristics. We show that facile linking of peptides together using different lengths of spacers and multivalency leads to a tuning of affinity and kinetics. Notably, increased length of spacers in divalent systems led to significantly increased affinities. Making multimers influenced also kinetic aspects of surface competition. Additionally, the multivalent peptides were applied as surface functionalization components for a colloidal form of DLC. The work suggests the use of a set of linking systems to screen parameters for functional optimization of selected material-specific peptides.

The structural basis for function in diamond-like carbon binding peptides.

The molecular structural basis for the function of specific peptides that bind to diamond-like carbon (DLC) surfaces was investigated. For this, a competition assay that provided a robust way of comparing relative affinities of peptide variants was set up. Point mutations of specific residues resulted in significant effects, but it was shown that the chemical composition of the peptide was not sufficient to explain peptide affinity. More significantly, rearrangements in the sequence indicated that the binding is a complex recognition event that is dependent on the overall structure of the peptide. The work demonstrates the unique properties of peptides for creating functionality at interfaces via noncovalent binding for potential applications in, for example, nanomaterials, biomedical materials, and sensors.

Formation of ceramophilic chitin and biohybrid materials enabled by a genetically engineered bifunctional protein.

A bifunctional protein composed of a highly negatively charged oyster shell protein and a chitin-binding domain enabled the formation of biohybrid materials through non-covalent surface modification of chitin nanofibres. The results demonstrate that specific biomolecular interactions offer a route for the formation of biosynthetic materials.

Selection and characterization of peptides binding to diamond-like carbon.

Phage display was used to find peptides specific for amorphous diamond-like carbon (DLC). A set of putative binders was analyzed in detail and one sequence was found that functioned both as a peptide fused to the pIII protein in M13 phage and as a peptide fused to the enzyme alkaline phosphatase (AP). The dissociation constant of the peptide-AP fusion on DLC was 63nM and the maximum binding capacity was 6.8pmol/cm(2). Multiple ways of analysis, including phage titer, enzyme-linked immunosorbent assay, and ellipsometry were used to analyze binding and to exclude possible false positive results. DLC binding peptides can be useful for self-assembling coatings for modifying DLC in specific ways.

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.

Genetic engineering in biomimetic composites.

Composites represent a class of materials with properties that are obtained by combining the functions of different components. Combining soft and stiff components without losing toughness is typically very difficult with current synthetic tools. There are many natural materials for which this problem has been solved. Examples such as wood and seashells have inspired many scientists to seek tougher, stronger and lighter materials. This review describes how genetic engineering can help in building new composites with better properties. Specifically, we emphasize that functional molecules can be engineered by following the design principles of natural composite materials. This field is emerging but has already shown promising results and much progress in the next few years is expected.

Engineering of a redox protein for DNA-directed assembly.

Engineered enzyme conjugate of the small laccase enzyme from Streptomyces coelicolor and zinc finger DNA binding domain from Zif268 is demonstrated to bind double stranded DNA in a site specific manner while retaining enzymatic activity.

Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold.

UNLABELLED: The de novo engineering of new proteins will allow the design of complex systems in synthetic biology. But the design of large proteins is very challenging due to the large combinatorial sequence space to be explored and the lack of a suitable selection system to guide the evolution and optimization. One way to approach this challenge is to use computational design methods based on the current crystallographic data and on molecular mechanics. We have used a laccase protein fold as a scaffold to design a new protein sequence that would adopt a 3D conformation in solution similar to a wild-type protein, the Trametes versicolor (TvL) fungal laccase. Laccases are multi-copper oxidases that find utility in a variety of industrial applications. The laccases with highest activity and redox potential are generally secreted fungal glycoproteins. Prokaryotic laccases have been identified with some desirable features, but they often exhibit low redox potentials. The designed sequence (DLac) shares a 50% sequence identity to the original TvL protein. The new DLac gene was overexpressed in E. coli and the majority of the protein was found in inclusion bodies. Both soluble protein and refolded insoluble protein were purified, and their identity was verified by mass spectrometry. Neither protein exhibited the characteristic T1 copper absorbance, neither bound copper by atomic absorption, and neither was active using a variety of laccase substrates over a range of pH values. Circular dichroism spectroscopy studies suggest that the DLac protein adopts a molten globule structure that is similar to the denatured and refolded native fungal TvL protein, which is significantly different from the natively secreted fungal protein. Taken together, these results indicate that the computationally designed DLac expressed in E. coli is unable to utilize the same folding pathway that is used in the expression of the parent TvL protein or the prokaryotic laccases. This sequence can be used going forward to help elucidate the sequence requirements needed for prokaryotic multi-copper oxidase expression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9080-9) contains supplementary material, which is available to authorized users.