Trichoderma reesei -homeen hydrofobiiniproteiinien itsejärjestäytyminen

Hydrophobins are small surface active proteins that are produced by filamentous fungi. The surface activity of hydrophobin proteins leads to the formation of a film at the air-water interface and adsorption to surfaces. The formation of these hydrophobin films and coatings is important in many stages of fungal development. Furthermore, these properties make hydrophobins interesting for potential use in technical applications. The surfactant-like properties of hydrophobins from Trichoderma reesei were studied at the air-water interface, at solid surfaces, and in solution. The hydrophobin HFBI was observed to spontaneously form a cohesive film on a water drop. The film was imaged using atomic force microscopy from both sides, revealing a monomolecular film with a defined molecular structure. The use of hydrophobins as surface immobilization carriers for enzymes was studied using fusion proteins of HFBI or HFBII and an enzyme. Furthermore, sitespecifically modified variants of HFBI were shown to retain their ability to selfassemble at interfaces and to be able to bind a second layer of proteins by biomolecular recognition. In order to understand the function of hydrophobins at interfaces, an understanding of their overall behavior and self-assembly is needed. HFBI and HFBII were shown to associate in solution into dimers and tetramers in a concentration-dependent manner. The association dynamics and protein-protein interactions of HFBI and HFBII were studied using Förster resonance energy transfer and size exclusion chromatography. It was shown that the surface activity of HFBI is not directly dependent on the formation of multimers in solution.Hydrofobiinit ovat rihmasienten tuottamia pieniä pinta-aktiivisia proteiineja. Pinta-aktiivisina molekyyleinä hydrofobiiniproteiinit muodostavat kalvon ilman ja veden rajapinnalle sekä kiinnittyvät kiinteille pinnoille. Näiden kalvojen ja pinnoitteiden muodostuminen on tärkeää monissa rihmasienen yksilönkehityksen vaiheissa. Lisäksi nämä ominaisuudet tekevät hydrofobiineista kiinnostavia molekyylejä teknisiin sovelluksiin. Trichoderma reesei -homeen tuottamien HFBI- ja HFBII-hydrofobiinien pintaaktiivisuusominaisuuksia tutkittiin ilman ja veden rajapinnalla, kiinteillä pinnoilla sekä liuoksessa. HFBI-hydrofobiinin havaittiin muodostavan yhtenäisen kalvon vesipisaran pinnalle. Kalvon molemmat puolet kuvattiin atomivoimamikroskoopilla ja osoitettiin, että kalvo on muodostunut yhdestä hydrofobiinimolekyylikerroksesta, jolla on hyvin järjestäytynyt molekyylitason rakenne. Entsyymien kiinnittämistä pinnoille hydrofobiinien avulla tutkittiin käyttäen HFBI:n tai HFBII:n ja mallientsyymin fuusioproteiineja. Lisäksi osoitettiin, että kohdennetusti muokattu HFBI-muunnos säilyttää kykynsä itsejärjestäytyä rajapinnoilla ja kykenee sitomaan hydrofobiini-kalvon päälle toisen proteiinikerroksen käyttäen biomolekulaarista tunnistusta. Ymmärtääksemme paremmin hydrofobiinien pinta-aktiivisuusominaisuuksia on tarpeen tuntea hydrofobiinien yleisiä ominaisuuksia ja itsejärjestäytymistä. HFBI sekä HFBII yhdistyvät vesiliuoksessa dimeereiksi ja tetrameereiksi, ja reaktio on riippuvainen proteiinien pitoisuudesta. Liuos-multimeerien muodostumisen dynamiikan ja proteiinien välisten vuorovaikutusten tutkimiseen käytettiin Försterresonanssienergiansiirtomenetelmää sekä geelisuodatuskromatografiaa. Tutkimuksessa osoitettiin, että HFBI:n pinta-aktiivisuus ei ole suoraan riippuvainen liuosmultimeerien muodostumisesta

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

We thank C. Li from the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany, for help during synchrotron measurements at the μSpot beamline at BESSY at the Helmholtz-Zentrum Berlin für Materialien und Energie in Berlin, Germany. We acknowledge the provision of facilities and technical support by Aalto University at the OtaNano Nanomicroscopy Center (Aalto-NMC). This work was supported by the Academy of Finland project 348628, the Jenny and Antti Wihuri Foundation (Centre for Young Synbio Scientists), and the Academy of Finland Center of Excellence Program (2022–2029) in Life-Inspired Hybrid Materials (LIBER) project number 346106, and by internal funding from the VTT Technical Research Center of Finland Ltd. We also acknowledge the Dutch Research Council (NWO, domain Applied and Engineering Sciences: MYCOAT project number 18425) and the Horizon 2020 programs of the European Union (FUNGAR; project 58132 and iNEXT-Discovery, project 871037) for NMR studies. Furthermore, the high-field NMR experiments were supported by uNMR-NL, the National Roadmap Large-Scale NMR Facility of the Netherlands (NWO grant 184.032.207), and the uNMR-NL grid (NWO grant 184.035.002).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.Peer reviewe

The dynamics of multimer formation of the amphiphilic hydrophobin protein HFBII

This work wassupported by the Academy of Finland through its Centres of Excel-lence Programme (2014–2019) and under Projects No. 259034 and264493.Hydrophobins are surface-active proteins produced by filamentous fungi. They have amphiphilic structures and form multimers in aqueous solution to shield their hydrophobic regions. The proteins rearrange at interfaces and self-assemble into films that can show a very high degree of structural order. Little is known on dynamics of multimer interactions in solution and how this is affected by other components. In this work we examine the multimer dynamics by stopped-flow fluorescence measurements and Förster Resonance Energy Transfer (FRET) using the class II hydrophobin HFBII. The half-life of exchange in the multimer state was 0.9 s at 22 °C with an activation energy of 92 kJ/mol. The multimer exchange process of HFBII was shown to be significantly affected by the closely related HFBI hydrophobin, lowering both activation energy and half-life for exchange. Lower molecular weight surfactants interacted in very selective ways, but other surface active proteins did not influence the rates of exchange. The results indicate that the multimer formation is driven by specific molecular interactions that distinguish different hydrophobins from each other.Peer reviewe

Genetically engineered protein based nacre-like nanocomposites with superior mechanical and electrochemical performance

The molecular engineering of proteins at the atomistic scale with specific material binding units and the introduction of designed functional-linkers provides a unique approach to fabricate genetically modified high performance and responsive biomimetic composites. This work is inspired by a tough biological material, nacre, which possesses a hierarchical 'brick-mortar' architecture containing multifunctional soft organic molecules, which plays a significant role in improved mechanical properties of composites. A bio-inspired composite, using a resilin-based hybrid protein polymer with selective binding motifs for reduced graphene oxide (RGO) and nanofibrillated cellulose (NFC), was developed. The adhesive and elastic domains of fusion proteins show a synergistic effect with improvement in both the strength and toughness of synthetic nacre. We observed that the hybrid protein could act as a spacer molecule tuning the ion sorption and transport across the inter-layers of NFC/RGO depending on the processing conditions. Interestingly, the protein complexed freestanding solid-state films showed negligible internal resistance and improved supercapacitance suitable for flexible electronic devices. The protein-mediated binding of NFC and RGO reduces the resistance arising from poor electrode/electrolyte interfaces, which is difficult to achieve through conventional routes. The current biosynthetic route for engineering proteins provides a novel prospect to develop materials programmed with desired properties, depending on target applications.Peer reviewe