Advanced optical techniques to study biomolecular aggregation processes
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Alzheimer’s disease (AD) is characterised by a series of biomolecular aggregation events, which include the formation of neurotoxic protein structures composed of the β-amyloid (Aβ) peptide. In this thesis, fluorescence self-quenching (FSQ) between fluorescently-labelled peptides is introduced as a strategy for detecting and characterizing Aβ aggregates in solution, and for overcoming limitations associated with conventional methods. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, the fluorescence response of HiLyte Fluor 555-labelled Aβ peptides is characterised to demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, N-terminal tagging of β-amyloid peptides is shown to not alter the self-assembly kinetics or the resulting aggregated structures. When performed in Förster resonance energy transfer (FRET) format, this method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid self-assembly. The ability of FSQ-based methods to monitor the inhibition of Aβ aggregation by model test compounds including the small heat shock protein (Hsp), the amyloid-binding alcohol dehydrogenase protein (ABAD) and bovine serum albumin (BSA) is also demonstrated. Given that Aβ is formed within the cell membrane and is known to induce its disruption, sophisticated single-molecule fluorescence spectroscopy methods were developed to quantify membrane dynamics induced by the presence of disrupting agents, such as Aβ and detergents. The solubilisation dynamics of single liposomes induced by the non-ionic surfactant Triton-X 100 (TX-100) were studied in real-time. Using this approach, the swelling and permeabilization steps of the solubilisation process were unambiguously separated within single FRET trajectories, and their kinetic details as a function of Triton-X 100 and presence of cholesterol within the membrane structure were examined. Finally, single-molecule stepwise-photobleaching techniques were employed to study the effect of Aβ oligomers interacting with supported-lipid bilayers, establishing a platform from which to investigate how the presence of a membrane layer affects Aβ oligomerization at the level of individual molecules. Overall, the fluorescence-based strategies for amyloid- and liposome-sensing presented in this work bridges the gap between current morphology-specific techniques and highly-specialized single-molecule methods to provide a biophysical toolbox to investigate the changes in structure, size and molecular interactions accompanying the amyloid aggregation pathway and for the screening of novel therapeutic and diagnostic agents.
Thesis, PhD Doctor of Philosophy
Embargo Date: 2023-03-18
Embargo Reason: Thesis restricted in accordance with University regulations. Electronic copy restricted until 18th March 2023
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