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.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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Proteins and peptides can convert from their native form into insoluble highly ordered fibrillar aggregates, known as amyloid fibrils. The process of fibrillogenesis is implicated in the pathogenic mechanisms of many diseases and, although mature fibrils are well characterised by a plethora of biophysical techniques, the initiation and early steps remain, to date, ambiguous. Mass spectrometry can provide invaluable insights into these early events as it can identify the low populated and transient oligomeric species present in the lag phase by their mass to charge ratio. Recent evidence has shown that oligomers formed early in the aggregation process are cytotoxic and may additionally be central to the progression of diseases associated with amyloid fibril presence. The hybrid technique of ion mobility mass spectrometry can be employed to provide conformational details of monomeric and multimeric species present and elucidate the presence of oligomers which possess coincident mass to charge ratios. Molecular modelling, in conjunction with experimental results, can suggest probable monomeric and oligomeric structural arrangements. In this thesis three aggregating systems are investigated: amyloidogenic transthyretin fragment (105-115), insulin and two Aβ peptides. Initially amyloidogenic endecapeptide transthyretin (105-115) is studied as it has been widely utilised as a model system for investigating amyloid formation due to its small size. Secondly insulin, a key hormone in metabolic processes, is investigated as extensive research has been carried out into its aggregation into amyloid fibrils. The formation of insulin amyloid fibrils rarely occurs in vivo; however localised amyloidosis at the site of injection and the aggregation of pharmaceutical insulin stocks present problems. Thirdly the aggregation of A β peptides Aβ (1-40) and Aβ (1-42) and their interactions with an aggregation inhibitor, RI-OR2, are characterised. A (1-42), although less commonly produced in vivo, is more cytotoxic and has a faster aggregation mechanism than Aβ (1-40). Both Aβ peptides are implicated in the aetiology of Alzheimer’s disease whilst RI-OR2 has been reported to prevent the production of high molecular weight oligomers, with particular suppression of Aβ (1-42) aggregation.

Master of Science / Department of Physics / Jeremy Schmit / In this work, we present a model for the kinetics of amyloid fibril aggregation. In the model we mapped the process of Hydrogen bond (H-bond) formation and breakage to a random-walk. we captured the effect of side chains using position dependent H-bonds free energies which allows us to calculated the residence time for different binding alignments with the fibril. The residence time can be compared to the diffusion-limited attachment rate to give net aggregation stability. This stability increases exponentially with increasing number of bonds or binding energy in homopolymer chains, however for chains with patterned sequences, the residence time shows strong effects of the binding alignment. Using the residence time for uniform structures combined with estimate of the diffusion rate, we modeled and simulated the kinetics of amyloid aggregation. Results of the simulations gives the bond energies and concentrations required for the onset of growth of aggregates.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2014. / Alzheimer’s Disease (AD) is characterized by neurofibrillary tangles, senile plaques and neuronal loss. Although the mechanisms underlying Amyloid beta 42 (Aβ42) neurotoxicity have not been firmly established, it is proposed that the neuronal loss is elicited through associations with cell surface receptors. The cellular prion protein (PrPc) has been identified as an Aβ42 receptor and as a regulator of the amyloidogenic cleavage pathway. As Aβ42 shares common binding partners with the 37kDa/67kDa laminin receptor (LRP/LR), including PrPc, we investigated whether these proteins interact and assessed the pathological significance of this association. LRP/LR was found to co-localize with Aβ on the cell surface. The occurrence of FRET suggested that an interaction between LRP/LR and Aβ indeed exists at the cell surface. Furthermore, pull down assays and Aβ-specific ELISAs demonstrated that LRP/LR forms a physical association with endogenously shed Aβ, thereby verifying the physiological relevance of this association. Antibody blockade by IgG1-iS18 and shRNAmediated downregulation of LRP/LR significantly enhanced cell viability and proliferation and decreased apoptosis in cells co-treated with Aβ42 when compared to cells incubated with Aβ42 alone. In addition, antibody blockade and shRNA-mediated downregulation of LRP/LR significantly impeded Aβ42 internalization. These results suggest that LRP/LR acts as an internalization receptor for Aβ42 and may thereby contribute to the cytotoxicity of the neuropeptide by facilitating intracellular Aβ42 accumulation and aggregation - which has consequences for cell proliferation and may promote apoptosis. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for Alzheimer’s Disease treatment.

Thesis advisor: Daniel A. Kirschner / In the present study, the ability of small compounds to inhibit the fibrillogenesis of beta-amyloid 12-28 was explored. Beta-amyloid 12-28 is a synthetic fragment of Alzheimer's beta-amyloid, which contains the core hydrophobic residues thought to be significant for fiber formation. Using x-ray diffraction, preliminary screening of over sixteen compounds was performed. Cotinine, PTI-00703®, and tetracycline were chosen because of their ease of solubility, the effect on the coherent domain size of the beta-crystallite subunit in the presence of chosen small molecules as shown by x-ray diffraction, as well as their presence in previously published literature. This conformational-driven inhibition of fibrillogenesis was explored in the current research using circular dichroism spectroscopy and x-ray diffraction. Circular dichroism spectroscopy revealed the nascent beta-sheet structure of beta-amyloid12-28 when first dissolved and only cotinine, out of all three inhibitors, was able to shift the equilibrium away from the fibrillogenic beta-sheet structure toward a random coil secondary structure after 36 hours of incubation. X-ray diffraction in this study demonstrated no change in hydrogen bond spacing at ~4.7Å and intersheet spacing at ~10-12Å both alone and in the presence of all small molecules. With increasing concentration of inhibitor, however, the widths of these reflections increased, indicating a decrease in the coherent domain size. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.