The current landscape of nanotechnology has focussed attention on materials that self-assemble. The search for such materials has unsurprisingly led to the biological world, where functional nanoscale biomolecular assemblies are in abundance. Amyloid fibrils are one such self-assembling biological structure, formed when native proteins misfold into insoluble fibrous quaternary structures. This research has explored the use of amyloid fibrils formed from waste proteins, namely crude crystallin proteins from fish eye lenses, as biological nanowires. The use of amyloid fibrils as nanowires was investigated by examining the ability to control their dimensions and arrangement, along with analysis of their properties, such as stability and conductivity. TEM and AFM studies on the model amyloid forming protein, bovine insulin, showed that a number of fibril length distributions can be achieved, by systematically altering fibril growth and storage conditions. Although the same set of conditions cannot be directly applied to crystallin fibrils, these fibrils can also be produced on a range of length scales. Amyloid fibrils can be manipulated and aligned in a controlled manner by dielectrophoresis; this tool could later be used to incorporate amyloid fibrils into a biosensing or bioelectronics device. Dielectrophoresis was also used to immobilise crystallin fibrils between electrode pairs, in order to investigate the conductivity of small numbers of fibrils. These experiments complemented work carried out on the conductivity of amyloid fibril networks, using fabricated interdigitated electrodes. In the unmodified state, amyloid fibrils formed from bovine insulin, fungal hydrophobins, and crude crystallins were all shown to have low conductivity, with current values in the range of 10⁻⁸–10⁻¹⁰ A recorded at bias voltages of 0–2 V. Amyloid fibrils were used as a template for the synthesis of conductive nanowires, by modification with the conducting polymers polyaniline and polypyrrole, increasing conductivity by one and four orders of magnitude respectively. The functionalisation of fibrils with glucose oxidase enabled the creation of a very simple glucose sensing device. This device, consisting of a gold electrode modified with the glucose oxidase functionalised fibrils, showed an electrochemical response in the presence of glucose and the mediator FcOH. Future work is necessary to optimise the use of amyloid fibrils in this way; however, this study confirms a role for amyloid fibrils from a low cost source in bionanotechnology.

Amyloid fibrils are a misfolded state formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them an excellent target for enzyme immobilisation and their strength, stability and nanometre size are attractive features for exploitation in the creation of new bionanomaterials. The aim of this thesis was to functionalise amyloid fibrils by conjugation to glucose oxidase (GOD). GOD is a relatively stable glycoprotein that catalyses the oxidation of glucose and the release of hydrogen peroxide. The consumption of glucose can be measured to assess glucose levels, and the release of hydrogen peroxide is cytotoxic to cells and is thus an effective antibacterial agent. Three methods of attachment were used: cross-linking using glutaraldehyde, periodate oxidation of the glycoprotein shell, and cross-linking using glutaraldehyde following deglycosylation. GOD retained activity upon attachment by all three methods. These attachment methods were assessed using electrophoresis, centrifugation, sucrose gradient centrifugation and TEM. Gel electrophoresis indicated a high degree of cross-linking and TEM showed no significant change of fibril morphology upon cross-linking. Centrifugation experiments suggested a non-covalent interaction was occurring between amyloid fibrils and GOD, and a covalent attachment was occurring upon addition of glutaraldehyde. Sucrose gradient centrifugation provided increased separation of cross-linked material compared to other separation methods, and showed greater cross-linking to crystallin amyloid fibrils than insulin fibrils. Cross-linking native GOD using glutaraldehyde was chosen for further experiments, as it was found to be most effective for GOD attachment to amyloid fibrils. The resulting functionalised enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The distribution of the functionalised fibrils through the film was characterised using SEM and confocal microscopy, where film components were found to be unevenly dispersed. The antibacterial effect of the functionalised film was then tested on E. coli and the antifungal effect of the film was tested on Fusarium, Rhizopus and Penicillium. Growth of E. coli was inhibited around functionalised film circles, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. However, no growth inhibition of fungal species was observed. This work is of significance as it demonstrates the ability to convert a waste material, bovine lens crystallin, to high value protein nanofibres and incorporate functionality via GOD attachment. The incorporation of the GOD-functionalised amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalised amyloid fibril scaffold. Future development of this model system has the potential to lead to the production of a novel biomaterial for use in food packaging due to the antimicrobial properties of GOD.

The aim of this thesis is to investigate and better understand the mechanisms of protein self-assembly. Specifically, I study three protein systems which form morphologically and structurally distinct brillar protein aggregates. The first of these studies is concerned with the self-assembly of amyloid brils formed from bovine insulin. Amyloid brils are associated with human diseases such as Alzheimers Disease and type-2 diabetes, and are also garnering interest in biomaterial applications. Fragmentation-dominated models for the self-assembly of amyloid brils have had important successes in explaining the kinetics of amyloid bril formation but predict bril length distributions that do not match experimental observations. Here I resolve this inconsistency using a combination of experimental kinetic measurements and computer simulations. I provide evidence for a structural transition demarcated by a critical bril mass concentration, or CFC, above which fragmentation of the brils is suppressed. Our simulations predict the formation of distinct bril length distributions above and below the CFC, which I confirm by electron microscopy. These results point to a new picture of amyloid bril growth in which structural transitions that occur during self-assembly have strong effects on the final population of aggregate species with small, and potentially cytotoxic, oligomers dominating for long periods of time at protein concentrations below the CFC. I further show that the CFC can be modulated by environmental conditions, pointing to possible in vivo strategies for controlling cytotoxicity. I probe the structural nature of the transition by performing small angle neutron scattering. Secondly, I study the formation of amyloid-like brils from the protein ovalbumin. I undertake kinetic experiments of self-assembly and find two key features emerge: the lack of a lag time and the existence of a slow growth regime in the long-time limit. I observe, using TEM, that these brils are worm-like in nature and form closed-loops. I find the growth kinetics are intimately connected to this particular morphology. I present a simple kinetic model which captures the features of the kinetics found in experiments by incorporating end-to-end association of brils. I comment on the ramifications this type of amyloid bril assembly may have on oligomeric toxicity. Thirdly, the DNA-mimic protein ocr is highly charged (-56e at pH 8) and forms non-amyloid brillar assemblies at very high ammonium sulphate concentrations (3.2M). The fact that ocr forms translucent brillar gels at such high salt concentrations is extremely unique. Typically under such high salt conditions, non-specific amorphous aggregates are formed. In order to better understand the mechanism of why ocr forms specific bril aggregates, I used variants of the wile-type protein in which extensive regions of surface have been removed or modified. The structural characteristics of gels formed from the variants were probed using microrheological techniques. I find that non-specific electrostatic charge screening plays an important role in ocr aggregation. However, I also locate a potentially important α-helical region which may play a part in establishing specific interactions so that ocr may form ordered brillar assemblies.

Protein nanofibres, commonly known as amyloid fibrils, are emerging as potential biological nanomaterials in a number of applications. Protein nanofibres are a highly ordered insoluble form of protein, which results when a normally soluble protein aggregates via a self-association process. However, researchers are currently faced with several challenges such as finding a cheap source of proteins that can be obtained without expensive purification and optimizing a scalable method of the manufacturing of protein nanofibres. This thesis has identified crude mixtures of fish lens crystallins as a cheap protein source and has optimized methods for large scale production of protein nanofibres of varying morphologies. Results show that by varying the conditions of fibre formation, individual protein fibres can be used as building blocks to form higher order structures. This ability to control the morphology and form higher ordered structures is a crucial step in bottom up assembly of bionanomaterials and opens possibilities for applications of protein nanofibres. The method of formation of protein nanofibres was optimized on a bench scale (1.5 mL Eppendorf tubes) and successfully scaled-up to 1 L volume. For larger scale-up volume (i.e. greater than 10 ml), internal surface area was important for the formation of protein nanofibres. The crude crystallin mixture prepared at 10 mg/mL was heated at 80oC in the presence of 10% v/v TFE at pH 3.8 for 24 hours and stored for an additional of 24 hours at room temperature for storage process. Aggregation and precipitation of proteins were observed as the protein solution was added to the pre-heated TFE. The resulting protein nanofibres were characterised using ThT dye binding, TEM and SEM. The TEM images show a network of long and criss-crossing protein nanofibres with individual fibres of approximately 10 to 20 nm in diameter and 0.5 to 1 μm long. These protein nanofibres were prepared in 1 mL centrifuge tubes and were left on the laboratory bench at room temperature. After 5 months, fresh TEM grids of the sample were prepared and visualized using TEM. Interestingly, TEM images show that a number of individual fibres had self-assembled in an intertwining fashion to form large bundles and higher order structures containing bundles of nanofibres up to 200 nm thick.

Many proteins are known to actively interact with biological, as well as inorganic and synthetic surfaces that are widely used in nano- and bio-technology as biosensing platforms and in tissue engineering. Amyloid fibrils are insoluble protein aggregates in beta-sheet conformation that are implicated in at least 20 diseases for which no cure is currently available. The molecular mechanism of fibril formation, as well as the mechanism of fibril clusters interacting with lipid membrane surfaces is currently unknown. The lipid membrane surface has a complex biochemical composition and is also electrostatically non-homogeneous. Currently, the experimental data available for amyloid fibril formation both on lipid and artificial surfaces is limited. The goal of our study is to investigate how the physical properties of the surfaces affect binding of amyloid peptides and affect the fibril formation. We seek to elucidate the effect of electrostatic interactions of amyloid peptides with surfaces using Atomic Force Microscopy (AFM) and Kelvin probe force microscopy (KPFM). We show using KPFM that electrostatic domains readily form within biological systems such as lung surfactant and lipid monolayers. We compared three different implementations of KPFM to demonstrate that frequency modulated (FM-) KPFM provides significant advantages over other modes. We also present a study of Amyloid beta (1-42) fibril formation on model surfaces, which are uniformly charged or possess periodicity of charges and hydrophobic functionality based on thiol self-assembly. Effect of membrane composition, surface charge, and presence of steroids will be discussed.

No description available.

Self-assembly of biomolecules into beta-sheet structures can be applied in the creation of nano-materials with novel electrical, optical, catalytical, or/and mechanical characteristics. This work was directed towards the construction of nano-derivatives based on amyloid fibrils forming proteins (Abeta40 peptide, a-Synuclein (a-Syn), equine lysozyme (EL)). Such nanostructures can be used to produce nanoscale functional systems. Herein, different mutant and hybrid proteins, which were able to form fibrillar structures, were constructed and the properties of fibrils were investigated. Designed cysteine mutants of Abeta40 and a-Syn can be modified through thiol group of cysteine. Herein, for the first time, it was demonstrated that a-Syncys141 fibrils could be modified with biotin and gold nanoparticles with neutravidin molecules. Hybrid proteins of Abeta40 or a-Syn and other non-amyloid proteins were designed on purpose to obtain fibrils with active functional non-amyloid proteins. Under appropriate conditions, these proteins aggregated into beta-sheet structures. Hybrid protein of streptavidin and Abeta40 formed a net-like fibrillar structure, and streptavidin was active. For the first time it was described the production of recombinant EL in E. coli. Moreover, active EL can form fibrils, which are similar to the fibrils formed by native EL. Constructed novel hybrids and mutants that are able to form amyloid fibrils, can be applied for the creation of functionalized nanodevices... [to full text] / Savitvarkės biomolekulės, gebančios formuoti beta-klosčių struktūras, gali būti pritaikomos nanomedžiagų su naujomis elektrinėmis, optinėmis, katalitinėmis ir/ar mechaninėmis savybėmis, kūrimui. Šiame darbe buvo siekiama kurti nanodarinius, grįstus amiloidines fibriles formuojančiais baltymais (Abeta40 peptidas, a-sinukleinas (a-Syn), kumelės pieno lizocimas (EL)), kurie būtų pagrindas nano dydžio funkcinių sistemų gamybai. Šiam tikslui buvo sukonstruoti mutantiniai ir hibridiniai baltymai bei tiriamos jų fibrilinių struktūrų savybės. Sukurti Abeta40 ir a-Syn cisteino mutantai, kurie gali būti modifikuojami per cisteino tiolinę grupę. Pirmą kartą buvo pademonstruotas a-Syncys141 baltymo fibrilių modifikavimas biotinu ir aukso nanodalelėmis su neutravidinu. Sukonstruoti hibridiniai baltymai, kurie sudaryti iš Abeta40 ar a-Syn bei GDH, streptavidino ir hidrofobino. Buvo tikimasi, kad tokie baltymai formuos fibrilines struktūras, o funkciniai baltymai bus aktyvūs. Esant atitinkamoms sąlygoms, šie baltymai agregavo suformuodami skirtingos morfologijos beta-klostines struktūras. Streptavidino ir Abeta40 hibridinis baltymas formavo fibrilinę struktūrą – tinklą, o streptavidinas buvo aktyvus. Šiame darbe pirmą kartą aprašoma rekombinantinio EL gamyba E. coli ląstelėse. Aktyvus EL gali formuoti panašias į natyvaus EL fibrilines struktūras. Sukonstruoti nauji hibridiniai ir mutantiniai baltymai, gebantys formuoti amiloidines fibriles, yra geras pagrindas, kuriant funkcionalizuotus... [toliau žr. visą tekstą]

Savitvarkės biomolekulės, gebančios formuoti beta klosčių struktūras, gali būti pritaikomos nanomedžiagų su naujomis elektrinėmis, optinėmis, katalitinėmis ir/ar mechaninėmis savybėmis, kūrimui. Šiame darbe buvo siekiama kurti nanodarinius, grįstus amiloidines fibriles formuojančiais baltymais (Abeta40 peptidas, a-sinukleinas (a-Syn), kumelės pieno lizocimas (EL)), kurie būtų pagrindas nano dydžio funkcinių sistemų gamybai. Šiam tikslui buvo sukonstruoti mutantiniai ir hibridiniai baltymai bei tiriamos jų fibrilinių struktūrų savybės. Sukurti Abeta40 ir a-Syn cisteino mutantai, kurie gali būti modifikuojami per cisteino tiolinę grupę. Pirmą kartą buvo pademonstruotas a-Syncys141 baltymo fibrilių modifikavimas biotinu ir aukso nanodalelėmis su neutravidinu. Sukonstruoti hibridiniai baltymai, kurie sudaryti iš Abeta40 ar a-Syn bei GDH, streptavidino ir hidrofobino. Buvo tikimasi, kad tokie baltymai formuos fibrilines struktūras, o funkciniai baltymai bus aktyvūs. Esant atitinkamoms sąlygoms, šie baltymai agregavo suformuodami skirtingos morfologijos beta klostines struktūras. Streptavidino ir Abeta40 hibridinis baltymas formavo fibrilinę struktūrą – tinklą, o streptavidinas buvo aktyvus. Šiame darbe pirmą kartą aprašoma rekombinantinio EL gamyba E. coli ląstelėse. Aktyvus EL gali formuoti panašias į natyvaus EL fibrilines struktūras. Sukonstruoti nauji hibridiniai ir mutantiniai baltymai, gebantys formuoti amiloidines fibriles, yra geras pagrindas, kuriant funkcionalizuotus... [toliau žr. visą tekstą] / Self-assembly of biomolecules into beta-sheet structures can be applied in the creation of nano-materials with novel electrical, optical, catalytical, or/and mechanical characteristics. This work was directed towards the construction of nano-derivatives based on amyloid fibrils forming proteins (Abeta40 peptide, a-Synuclein (a-Syn), equine lysozyme (EL)). Such nanostructures can be used to produce nanoscale functional systems. Herein, different mutant and hybrid proteins, which were able to form fibrillar structures, were constructed and the properties of fibrils were investigated. Designed cysteine mutants of Abeta40 and a-Syn can be modified through thiol group of cysteine. Herein, for the first time, it was demonstrated that a-Syncys141 fibrils could be modified with biotin and gold nanoparticles with neutravidin molecules. Hybrid proteins of Abeta40 or a-Syn and other non-amyloid proteins were designed on purpose to obtain fibrils with active functional non-amyloid proteins. Under appropriate conditions, these proteins aggregated into beta-sheet structures. Hybrid protein of streptavidin and Abeta40 formed a net-like fibrillar structure, and streptavidin was active. For the first time it was described the production of recombinant EL in E. coli. Moreover, active EL can form fibrils, which are similar to the fibrils formed by native EL. Constructed novel hybrids and mutants that are able to form amyloid fibrils, can be applied for the creation of functionalized nanodevices... [to full text]

The research field of conjugated polymers has grown due to the optical and electronic properties of the material, useful in applications such as solar cells and printed electronics, but also in biosensors and for interactions with biomolecules. In this thesis conjugated polymers have been used in two related topics; to detect conformational changes in proteins and to assemble the polymers with biomolecules into nanowires. Within biosensing, conjugated polymers have been used for detection of a wide range of biological events, such as DNA hybridization or enzymatic activity, utilizing both electronic and optical changes in the polymer. Here the focus has been to use the polymers as optical probes to discriminate between native and misfolded protein, as well as to follow the misfolding processes in vitro. The understanding and detection of protein misfolding, for example amyloid fibril formation, is a topic of growing importance. The misfolding process is strongly associated with several devastating diseases such as Alzheimer’s disease, Parkinson’s disease and Bovine Spongiform Encephalopathy (BSE). We have developed detection schemes for discrimination between proteins in the native or amyloid fibril state based on luminescent polythiophene derivatives. Through a synthesis strategy based on polymerization of trimer blocks rather than of monomers, polythiophene derivatives with higher optical signal specificity for amyloid-like fibrils were obtained. Self-assembly of nanowires containing conjugated polymers is a route to generate structures of unique opto-electrical characteristics without the need for tedious topdown processes. Biomolecules can have nanowire geometries of extraordinary aspect ratio and functionalities. The DNA molecule is the most well known and exploited of these. In this thesis work the more stable amyloid fibril has been used as a template to organize conjugated polymers. Luminescent, semi-conducting, conjugated polymers have been incorporated in and assembled onto amyloid fibrils. Using luminescence quenching we have demonstrated that the conjugated material can retain the electro-activity after the incorporation process. Furthermore, the amyloid fibril/conjugated polymer hybrid structures can be organized on surfaces by the means of molecular combing and soft lithography. In the process of generating self-assembled biomolecular nanowires functionalized with conjugated polymers, we have shown a new synthesis strategy for a water-soluble highly conducting polythiophene derivative. This material, PEDOT-S, has shown affinity for amyloid fibrils, but can also be very useful in conventional opto-electronic polymer-based devices.

In this thesis we present computer modelling studies that were implemented to investigate protein behavior in various environments causing their folding, unfolding and aggregation. Applications related to two important proteins - insulin and apolipoprotein C-II (ApoC-II) are presented. The use of atomistic simulation methodologies based on empirical force fields has enhanced our understanding of many physical processes governing protein structure and dynamics. However, the force fields used in classical modelling studies are often designed for a particular class of proteins and rely on continuous improvement and validation by comparison of simulations with experimental data. In Chapter 4 we present a comprehensive comparison of five popular force fields for simulation of insulin. The effect of each force field on the conformational evolution and structural properties of the protein is analysed in detail and compared with available experimental data. A fundamental phenomenon in nature is the ability of proteins to fold ab initio to their functional native conformation, also known as their biologically active state. Due to the heterogeneity and dimensionality of the systems involved, it is necessary to employ methodologies capable of accelerating rare events, specifically, configurational changes that involve the crossing of large free energy barriers. In Chapter 5, using the recently developed method BE-META we were able to identify the structural transitions and possible folding pathways of insulin. Another interesting phenomenon is the misfolding of proteins causing their aggregation, that may lead to formation of either amorphous compounds or structures of elongated-unbranched morphology known as amyloid fibrils. The deposition of amyloid fibrils in the human body may cause many debilitating diseases such as Alzheimer's and variant Creutzfeldt-Jakob diseases, thus making this field of research important and urgent. The human plasma protein apoC-II serves important roles in lipid transport, and it has been shown to form amyloid-like aggregates in solution. We have performed computational studies to investigate the effect of mutations, such as Met oxidation and the residue substitutions to hydrophobic Val and hydrophilic Gln, on dynamics of apoC-II(60-70) peptide. The conformation features relevant to the amyloidogenic propensities of the peptide were identified and presented in Chapter 6. The involvement of lipids at the various stages of development of amyloid diseases is becoming more evident in recent research efforts. In particular, micellar and sub-micellar concentrations have showed to have different effect on fibril growth and kinetics of native apoC-II and derived peptides. In Chapter 7 we investigated the influences of phospholipids at various concentrations on the structure of apoC-II(60-70) using MD and umbrella sampling methods. The molecular mechanisms of lipid effects on the peptide conformation and dynamics were identified. In Chapter 8 preliminary results on the structural stability of pre-formed oligomeric composites of apoC-II(60-70) peptide of different sizes and arrangements were also presented. The effects of mutation (oxidised Met, Met60Val and Met60Gln) on the most stable cluster was also investigated. To conclude, several ideas for continuation of research in the protein folding and aggregation field are discussed in the Future Work section of this thesis.