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Title: Fluorescence studies of the interactions of amyloid-β with metal ions and lipids
Author: Branch, Thomas
ISNI:       0000 0004 5920 6855
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Amyloid-β (Aβ) metal interactions promote aggregation and produce reactive oxygen species, hallmarks of Alzheimer's disease (AD). Oligomerisation of Aβ has been proposed to be modulated by copper and zinc ions. In environments of fast molecular turnover, such as the synaptic cleft, the kinetics of molecular interactions are important. To assess the roles of Cu²⁺ and Zn²⁺ in the early molecular events of AD, the kinetics of Aβ binding to both ions were determined. Metal binding was monitored using the quenching of a fluorescent dye covalently linked to Aβ by Cu²⁺, enabling measurements to be performed at physiologically relevant Aβ concentrations. The binding of monomeric Aβ to Cu²⁺ was nearly diffusion limited with a lifetime of a few seconds. Two forms of Aβ-Cu were found that interconverted, and at least two further Cu²⁺ ions could bind. The protonated form of Aβ-Cu was capable of dimerisation. Cu²⁺ assisted dimerisation is two orders of magnitude faster than without Cu²⁺. A metric was devised to measure the effectiveness of the removal of Cu²⁺ from Aβ-Cu by a ligand. The kinetics of Cu²⁺ binding to Aβ pre-bound to GM1 micelles were similar to unbound Aβ, however GM1 micelles protected the Aβ complex from Cu²⁺ binding ligands. Zn²⁺ binding to Aβ was two orders of magnitude slower than Cu²⁺, with a lifetime of tens of milliseconds. The binding of Zn²⁺ to Aβ-Cu, was a further three orders of magnitude slower. A reaction-diffusion simulation of the repeated release of neurometals into a synaptic cleft suggested that Aβ-Cu is two orders of magnitude more likely to form than Aβ-Zn under physiological conditions. This suggests that Cu²⁺ rather than Zn²⁺ is responsible for the dimerisation of Aβ in the synaptic cleft. The methodology applied here is applicable to determine the Cu²⁺ binding kinetics of other peptides or proteins.
Supervisor: Ying, Liming ; Barahona, Mauricio ; Barter, Laura Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available