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.

Amyloid fibrils are an insoluble, highly ordered, fibrous protein structure, which have increasingly been recognised as having bionanotechnology applications. Their ability to selfassemble allows a bottom-up approach to material design. Their nanometre dimensions affords them a high surface-to-volume ratio and their proteinaceous building blocks from which they are assembled allow for decoration with biomolecules and chemicals through amino acid residues. Amyloid fibrils are therefore a potential nanoscaffold for immobilisation of biomolecules. Immobilisation offers a solution to the problems associated with the use of enzymes in in vitro applications, by increasing their stability, reusability, and in some cases, enhancing catalytic activity. Nanosupports offer a high surface-to-volume ratio compared to classical planar 2-D supports, potentially affording them dramatic increases in immobilisation capacity. To investigate the potential of amyloid fibrils as a novel nanoscaffold, organophosphate hydrolase (OPH), cytochrome P450BM3 (P450BM3), green fluorescent protein (GFP), tobacco etch virus protease (TEV), and glucose oxidase (GOD) were immobilised in solution to the model amyloid fibril forming protein, bovine insulin. Covalently immobilised OPH was found to have a ~300 % increase in relative thermostability at 40 and 50 °C. P450BM3 was not successfully immobilised in its active state, most likely due to unfolding of the enzyme on the amyloid fibril surface. Covalently immobilised GFP retained full fluorescence and acted as a fluorescent protein tag. TEV was shown to have a physical interaction with the nanoscaffold and retain activity. GOD was immobilised and retained activity. Although not all proteins retained activity, a range of different protein structures were successfully immobilised onto the insulin amyloid fibril nanoscaffold. Attachment to the crystallin amyloid fibril nanoscaffold remains a work in progress due to the complexities associated with post-translational modifications of these fibrils. Crystallin amyloid fibrils were assembled on a surface for the first time. Their surface assembled structure was found to resemble spherulites, not previously seen before with crystallin amyloid fibrils. Bovine insulin amyloid fibrils were assembled on the surface of glass beads to increase the available surface area for biomolecule immobilisation. The surface assembled bovine insulin nanoscaffold was first functionalised with GOD, demonstrating that the nanoscaffold provides more surface area for biomolecule immobilisation, although in this case the increase was limited due to high non-specific binding of GOD to the unmodified glass surface. GFP was successfully employed as a fluorescent protein tag to assess the degree of nanoscaffold coverage, confirming the nanoscaffold affords the glass bead a greater surface area. Moreover, a reusable immobilised TEV protease-bead system was developed that was able to sequentially cleave the poly-histidine tags of three different proteins. In conclusion, bovine insulin amyloid fibrils have been shown to be a versatile nanoscaffold for the immobilisation of a range of biomolecules. The surface characteristics of the nanoscaffold allows for both covalent and physical immobilisation of biomolecules. Thus, amyloid fibrils have exciting potential in the creation of novel bionanotechnologies.

The inverse scattering problem is based on the scattering theory in physics, where measured data such as radiation from an object is used to determine the unique structure of the object in question. This approach has been widely successful in fields ranging from geophysics and medical imaging, to quantum field theory. In 1996 Henrik Flyvbjerg suggested that a similar approach could be used to study a reaction far from equilibrium of the self-assembly of a nucleation dependent biopolymer and, under certain conditions, uniquely determine the kinetics of the assembly. Here we use this approach to elucidate the unique structure of human islet amyloid polypeptide, also known as amylin, in-vitro. We use a systematic phenomenological analysis of the amount of monomer in fibril, of amylin, for various initial concentrations from an unstructured monomer pool. Using the assumption that nucleation is the rate-limiting step in fibril formation, we invoke mass action to develop our model. We find that the fibrillogenesis of amylin is well described by a nucleation dependent polymerization event that is characteristic of the sigmoidal shape of the reaction profile generated by our data. Furthermore, we find a second nucleation event is needed to accurately match model predictions to the observed data for the kinetic profiles of fibril formation, and the experimental length distributions of mature fibrils from in-vitro assays. This analysis allows for the theoretical determination of each step of assembly in the nucleation process. Specifically, we find the number of steps to nucleation, the size of each oligomer formed in the nucleation process, the nucleus size, and the elongation kinetics of fibrils. The secondary nucleation process is found to be a fibril dependent surface mediated nucleation event and is similar in reaction order to the primary nucleation step. Model predictions are found to be congruent with experimental assay results of oligomer populations and monomer concentration. We demonstrate that, a persistent oligomer formation is a natural and necessary consequence of nucleated fibril formation, given certain qualitative features of the kinetic profile of fibril formation. Furthermore, the modeling assumptions about monomer and fibril mass are in agreement with experiment.

Thesis (Ph.D.)--Tufts University, 2004. / Adviser: Eliana De Bernardez Clark. Submitted to the Dept. of Chemical Engineering. Includes bibliographical references (leaves 173-181). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph.D.) -- University of Adelaide, Dept. of Pathology, 1999. / Appendum pasted into front end-papers. Bibliography: leaves xiii-xliii.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 113-125). Also available in electronic version. Access restricted to campus users.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 14, 2009) Includes bibliographical references.

The inverse scattering problem is based on the scattering theory in physics, where measured data such as radiation from an object is used to determine the unique structure of the object in question. This approach has been widely successful in fields ranging from geophysics and medical imaging, to quantum field theory. In 1996 Henrik Flyvbjerg suggested that a similar approach could be used to study a reaction far from equilibrium of the self-assembly of a nucleation dependent biopolymer and, under certain conditions, uniquely determine the kinetics of the assembly. Here we use this approach to elucidate the unique structure of human islet amyloid polypeptide, also known as amylin, in-vitro. We use a systematic phenomenological analysis of the amount of monomer in fibril, of amylin, for various initial concentrations from an unstructured monomer pool. Using the assumption that nucleation is the rate-limiting step in fibril formation, we invoke mass action to develop our model. We find that the fibrillogenesis of amylin is well described by a nucleation dependent polymerization event that is characteristic of the sigmoidal shape of the reaction profile generated by our data. Furthermore, we find a second nucleation event is needed to accurately match model predictions to the observed data for the kinetic profiles of fibril formation, and the experimental length distributions of mature fibrils from in-vitro assays. This analysis allows for the theoretical determination of each step of assembly in the nucleation process. Specifically, we find the number of steps to nucleation, the size of each oligomer formed in the nucleation process, the nucleus size, and the elongation kinetics of fibrils. The secondary nucleation process is found to be a fibril dependent surface mediated nucleation event and is similar in reaction order to the primary nucleation step. Model predictions are found to be congruent with experimental assay results of oligomer populations and monomer concentration. We demonstrate that, a persistent oligomer formation is a natural and necessary consequence of nucleated fibril formation, given certain qualitative features of the kinetic profile of fibril formation. Furthermore, the modeling assumptions about monomer and fibril mass are in agreement with experiment. / Science, Faculty of / Mathematics, Department of / Graduate

Alzheimer’s disease (AD) is the most common form of dementia, with more than 25 million people worldwide suffering this progressive intellectual failure. The disease was first described by the German psychiatrist, Alois Alzheimer in 1907, and is characterised by the appearance of proteinaceous depositions (first isolated in 1984), which are comprised of insoluble amyloid-ß (Aß)-aggregates. Aβ is derived from the β-amyloid precursor protein from which it is generated by the action of two proteases. Initially it was assumed that the insoluble amyloid fibrils, which were easily detectable, mediated the observed toxicity although it was recognised that amyloid plaque number did not correlate well with the severity of dementia. However, further studies with synthetic and human-derived Aß provided strong evidence that soluble prefibrillar aggregates of Aß mediated the synaptic failure and loss of cognitive performance. In 2008 genetic evidence showed that the presence of soluble Aß-oligomers is sufficient to cause an AD-like dementia, which centres the oligomeric Aβ as the probable effector of synapse loss. Although a variety of assemblies have been described their meta-stability and technical limitations caused a controversial debate about aggregate related pathogenesis. Thus, this study aimed to establish a structure-activity relationship comparing different synthetic Aß-aggregates using biophysical methods to follow aggregation and to assess morphology, absolute MW and meta-stability of monomeric, oligomeric, protofibrillar and fibrillar Aß. However, interference with the aggregate equilibrium, by changing the ionic environment, can cause structural conversion of Aß-aggregates. Therefore, different Aß-aggregates were only compared in short-termed physiological settings i.e. neuronal binding and hippocampal neurotransmission. Herein, only prefibrillar aggregates bound to neurons and differentially impaired hippocampal neurotransmission either by inhibition of basal neurotransmission or NMDA-dependent long-term potentiation. In addition, changing the ionic environment provoked a structural conversion, which also changed the pathogenic mode of action. This study provides experimental evidence that different soluble Aß-aggregates are highly potent synaptotoxins, impairing neurotransmission by different mechanisms. Furthermore, solution-based biophysical characterisation and acute biological paradigms are crucial for differential characterisation of Aß-aggregates revealing that virtually similar aggregates can have opponent pathogenic effects; thus, morphology only does not explain observed pathogenicity.

The generation of amyloid-β (Aβ) peptides through proteolytic processing of the amyloid precursor protein (APP) is a key pathogenic event in Alzheimer’s disease (AD). Aβ generation begins with APP endocytosis, which is mediated by the endocytic YENPTY sequence located in the cytoplasmic tail of APP. Mints, a family of cytosolic adaptor proteins, directly bind to the YENPTY motif of APP and facilitate APP endocytosis and amyloidogenic processing. In addition, loss of any one of the three Mint proteins decreases Aβ production in aging mouse models of AD, supporting the hypothesis that the APP-Mint interaction may provide a novel therapeutic target to selectively reduce Aβ production in AD. Characterizing the biochemical and cellular dynamics of the APP-Mint interaction is critical for understanding Aβ generation. Thus, we generated Mint1 mutants that bind with high affinity (Mint1Y633A) or low affinity (Mint1Y549A/F610A) to APP. These Mint1 mutants exhibited profound alterations in cellular localization, APP endocytosis, and Aβ production. Therapeutically, we generated a novel cell-permeable APP mimetic peptide (APPMP) that interferes with the APP-Mint interaction. This APPMP was designed to outcompete endogenous APP binding, with a 46-fold improved affinity to Mint. Treatment of primary neurons from an AD mouse model with several cell permeable APPMP variants reduced Aβ production with minimal cellular toxicity, supporting Mints as a promising novel therapeutic target for AD. The PTB domain of Mint1 that mediates APP binding is autoinhibited by an adjacent C-terminal α-helix. However, the molecular mechanisms underlying the relief of Mint1 autoinhibition are unclear. Since post-translational modification is one mechanism for alleviating protein autoinhibition, and Mint1 is highly regulated by phosphorylation, we performed mass spectrometry and identified several Mint1 phosphosites. In addition, we found constitutively-active Src kinase, a kinase implicated in Mint phosphorylation, enhanced APP-Mint1 binding. These results suggest that Src kinase-mediated phosphorylation of Mint1 may relieve Mint1 autoinhibition and promote APP-Mint1 interaction. Overall, this work biochemically characterized the Mint-APP interaction and how it affects amyloidogenic processing, provided a proof of concept for targeting the APP-Mint1 interaction as an AD therapeutic target, and suggested a novel mechanism for the relief of Mint1 autoinhibition.