Hydrophobin Film Structure for HFBI and HFBII and Mechanism for Accelerated Film Formation

Peer reviewe

Molecular interactions of hydrophobin proteins with their surroundings

This thesis describes the properties of a group of proteins named hydro-phobins, which fulfil a variety of functions in the growth and function of filamentous fungi. Hydrophobins can be utilized as coatings/protective agents, in adhesion, in surface modifications and overall functions that require surfactant-like properties. This work is concentrated on the hy-drophobins HFBI, HFBII and HFBIII expressed by Trichoderma reesei. The aims of this study were to examine in what manner hydrophobins function when interacting with their surroundings and how their surroundings affect their function. Hydrophobins were shown strongly to adhere to surfaces of varying polarity and structure by self-assembly, governed by their amphiphilic nature, and to adsorb with different orientation on hydrophilic and hydrophobic surfaces. The proteins were shown to selectively recruit other proteins and molecules to a self-assembled amphiphilic film of hydrophobin. HFBI variants bound to a surface were shown to recruit T. reesei enzymes specifically depending on localized protein surface charge on the hydrophilic part of the protein, and HFBII adsorbed on nanoparticles was shown to bind layers of human plasma proteins in different manner when adsorbed on nanoparticles of varying polarity. Surface films formed by hydrophobins were shown to be highly elastic, and charged residues on the side of the proteins were shown to have a role in stabilizing the protein films formed. The surroundings in which the proteins exist were shown to also affect their function. Surfaces of varying polarity in the protein surroundings affected how they self-assemble, and hydrophobin multimer exchange in solution was shown to be governed by hydrophobic interactions and the multimer exchange behaviour was shown to be affected by other proteins and molecules. HFBII and HFBI were shown to interact in solution, altering multimer kinetics and thermodynamics considerably. Solution association methods, surface characterization analysis methods and size measurement techniques such as stopped-flow spectroscopy, quartz crystal microbalance with dissipation and differential centrifugal sedimentation were used. The results presented here show that hydrophobins function by selectively interacting with their surroundings assembled at various interfaces specifically recruiting other proteins and molecules and that the surroundings in which the proteins exist also affects their function in terms of multimer exchange behaviour and surface adhesion properties. The knowledge learned here regarding hydrophobins, show that these proteins can be specialized to function as highly selective self-assembling building blocks in applications such as biosensors and biocompatible coatings, and gives new insight in the growth and function of filamentous fungi

Hydrophobins: Biological application of fungal proteins

Hydrophobin protiens are unique to the fungal kingdome and have evolved to function in different roles during the growth and development of filamentous fungi. Due to the unique properties of these proteins to self-assemble into amphipathic monolayers at hydrophobic:hydrophlic interfaces, they can be found as biosurfactants, protective coatings and as primers to enhance surface adhesion. In recent decades there has been a significant development towards the applying these proteins to a range of different research fields, from food technology and surface coatings to drug delivery devices. However, the understanding of the mechanism in which these proteins undergo self-assemble at the interface is still lacking. In this project, I have combined high resolution imagaing techniques, such as AFM, TEM and TEM tomography to compare the substructure of different Class I hydrophobin rodlet films, and using colometric kinetic assays to delineate a model for the assembly mechanisms at the interface. With the information, I was able to reveal that the exposure of hydrophobin proteins to the surface interface is a determining factor. By altering the surface interface with additives, such as ethanol, it was possible to manipulate the hydrophobin film structure and physioproperties. This knowledge was used to successfully formulate a nanosupsension of hydrophobin with hydrophobic compounds, such as curcumin and Amphotericin B. This research project lays the foundation for the future development and refinement of hydrophobin-based technologies

Lateral interactions govern self-assembly of the bacterial biofilm matrix protein BslA

The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.</p

Lateral interactions govern self-assembly of the bacterial biofilm matrix protein BslA

The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.</p

Biopolymer-Based Nanoparticles for Drug Delivery

Nanotechnology can be used to modify drug delivery by various approaches. Bio-polymer based nanoparticles represent a well-established option to formulate drug delivery systems. New therapeutic compounds are often insoluble or poorly soluble in water, which is a major factor in causing irregular and insufficient absorption, and reduced bioavailability of the drug. Increased dissolution rate can be achieved by decreasing the particle size to nanometer range. Another common reason for the reduced efficacy of the therapeutic compounds is their poor delivery to the desired site of action. Advanced nanoparticle formulations can be used to provide controlled release profiles or they can be combined with ligands for targeted drug delivery. Considering the current needs to produce stable nanoparticle systems for the controlled and actively targeted drug release, the versatile group of biopolymers may offer these functionalities. One topic of this work was to set-up the electrospray apparatus for the production of poly(lactic acid) (PLA) drug nanoparticles. By utilizing electrospray, it was possible to produce spherical drug-loaded PLA-particles with approximately 200 to 800 nm diameters. The main benefits of the electrospray method were to control the particle size as well as the possibility to entrap both hydrophobic and hydrophilic drugs into the polymeric nanoparticles. Second topic of this work was to utilize amphiphilic proteins, hydrophobins, to provide a layer around the drug nanoparticles that can be functionalized by protein engineering techniques. Adsorption of the protein onto the particle surface restricted the particle growth after the nanoparticles were formed, and it also produced a layer around the hydrophobic drug that was possible to functionalize further. Hydrophobin-mediated nanoparticle synthesis was a fast and effective process that was also easy to up-scale. As third topic, nanofibrillar celluloses (NFCs) from various origins were studied as alternative biopolymer carriers for drug nanoparticles. The nanostructured cellulose matrix, an aerogel, prevented the aggregation of the hydrophobin coated nanoparticles during the freeze-drying and storage. Controlled drug release applications could be designed and enabled by utilizing the various modifications of NFC matrices. As a result of this thesis, knowledge about the versatile biopolymer-based materials was provided as a means to construct stable nanoparticle formulations that can offer versatile applications for pharmaceutical nanotechnology.Monet terapeuttisesti potentiaaliset molekyylit tulevat hylätyiksi jo lääkekehityksen alkumetreillä, koska heikon vesiliukoisuutensa takia lääkeaine ei tuottaisi elimistössä haluttua vaikutusta. Lisäksi monien lääkeaineiden toimiva, tehokas ja turvallinen annostelu edellyttää usein säädeltyä vapautumista lääkevalmisteesta. Lääkeaineesta ja kantajasta koostuvia nanokokoluokan partikkeleita voidaan soveltaa lääkkeenkuljettimina erilaisissa lääkkeenannostelun haasteissa. Nanoteknologisilla apukeinoilla voidaan parantaa ja säädellä lääkeaineen vapautumista elimistössä, jonka lisäksi nanopartikkeleissa käytetyn kantajamateriaalin avulla voidaan suojata herkkiä lääkeaineita elimistössä ennenaikaista hajoamista vastaan, sekä kohdentaa lääkkeen annostelu täsmällisemmin vaikutuspaikalleen. Kantajamateriaaleilta vaaditaankin sopivan kemiallisen koostumuksen lisäksi mahdollisuutta kuljetussysteemin rakenteiden ja vuorovaikutusten kontrollointiin. Useita biopolymeerejä pidetään varteenotettavina vaihtoehtoina lääkkeenkuljetussysteemeissä niiden biologisen yhteensopivuuden sekä hajoavuuden takia, mutta myös räätälöitävien ominaisuuksien ansiosta. Farmaseuttisten nanopartikkeleiden merkittävimpiin haasteisiin kuuluvat teollisen mittakaavan valmistukseen liittyvät ongelmat sekä käsittelyn ja säilytyksen aikainen fysikaalinen epästabiilisuus. Tämän tutkimuksen tärkein tavoite oli kehittää nanoteknologisia menetelmiä nanopartikkeleiden tuottamiseen sekä tutkia uusia biomateriaaleja lääkenanopartikkeliformulaatioiden kehityksessä. Kantajamateriaalien valinnalla pyrittiin lääkeaineen ja nanopartikkeleiden stabiilisuuden parantamiseen sekä lääkeaineen säädeltävään vapautumiseen formulaatiosta. Tavoitteena oli saavuttaa lääkeaineen nopea tai pitkitetty vapautuminen muodostuvista nanopartikkeleista tai nanokomposiittirakenteista. Lääkeaineiden kantajamateriaaleina käytettiin biologista alkuperää olevia polymeerimateriaaleja: polymaitohappoa, hydrofobiiniproteiineja sekä nanokuituista selluloosaa. Työssä kehitettiin sähkösumutusta menetelmänä tuottaa halutun kokoisia polymeerisiä, polymaitohappo (PLA) lääkeainenanopartikkeleita sekä hydrofiilisestä että hydrofobisesta lääkeaineesta. Lisäksi työssä kehitettiin menetelmä, jossa lääkkeellisiä nanopartikkeleita tuotettiin ohjaamalla molekyylien itsejärjestäytymistä käyttämällä amfifiilisiä proteiineja, hydrofobiineja, toiminnallisina apuaineina. Hydrofobiinit itsejärjestäytyivät muodostuvien nanopartikkeleiden pinnalle ohjaten lääkeainepartikkelin muodostusta, suojaten niitä aggregaatiolta sekä parantaen hydrofobisen lääkeaineen biologista hyötyosuutta elimistössä. Geneettisellä manipuloinnilla hydrofobiiniin voidaan fuusioida erilaisia toiminnallisia ryhmiä tai ligandeja, jolloin mm. nanopartikkeleiden aktiviinen kohdentaminen on mahdollista. Tässä työssä selluloosaan kiinnittyvän moduulin (cellulose binding domain, CBD), avulla nanopartikkelit saatiin liitettyä ei-kovalenttisesti nanokuituiseen sellulosaan. Erilaiset nanokuituiset selluloosat osoittautuivat lupaaviksi materiaaleiksi nanokomposittirakenteiden muodostajina. Kylmäkuivausprosessin aikana selluloosakuidut muodostivat huokoisia rakenteita suojaten nanopartikkeleita sekä prosessoinnin että säilytyksen aikana, mutta in vitro - tutkimusten perusteella ne myös kontrolloivat lääkeaineen vapautumista valmisteesta. Tutkimuksen tulosten perusteella käytetyt materiaalit sekä itsejärjestäytyvät systeemit antavat edellytykset toimivien nanopartikkelilääkeformulaatioiden kehitykseen