Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.570606
Title: Photophysical studies of amyloid aggregation
Author: Amaro, Mariana Manuela Salgado da Costa
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2011
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Abstract:
Fluorescent techniques are amongst the methods used to study the self assembly of proteins into oligomers, however, most of the reports in literature use extrinsic fluorophores to obtain information on the peptide system. The goal of this thesis is to demonstrate how the intrinsic Tyrosine fluorescence of the β-amyloid peptide can be used to monitor its own aggregation with, therefore, minimal interference on the peptide's native structure, its biochemistry and its spontaneous process of aggregation. Firstly, it is shown that the fluorescence of amino acid Tyrosine, naturally present in wild type β-amyloid, responds to the spontaneous aggregation of the peptide. This is achieved by performing time-correlated single-photon counting experiments during the process of the peptide's aggregation into amyloid fibrils. Through comparison with the well established Thioflavin T assay is also demonstrated that Tyrosine decay responds to changes caused by peptide aggregation well before the appearance of the characteristic β- sheet structures present in the fibrils. Then the use of β-amyloid's intrinsic Tyrosine amino acid as a sensor for the pre- brillar stages of aggregation is further tested and researched through a series of experiments with different initial β-amyloid concentrations. Patterns of consistent behaviour are found confirming that Tyrosine can act as a sensor for the formation of oligomers and kinetic information about the oligomerisation process is retrieved. Using this approach a comparison between the oligomerisation kinetics of the two most common variants of β-amyloid is performed. In the process of studying Tyrosine response to the peptide aggregation the accuracy of the discrete exponential decay model used to describe the decays is debated. A model-free analysis is used to study Tyrosine decay photophysics in the β-amyloid peptides throughout the process of aggregation. It is found that Tyrosine decay is a composition of four discrete decay components suggesting the existence of four rotameric forms of the amino acid in the Fluorescent techniques are amongst the methods used to study the self assembly of proteins into oligomers, however, most of the reports in literature use extrinsic uorophores to obtain information on the peptide system. The goal of this thesis is to demonstrate how the intrinsic Tyrosine uorescence of the β-amyloid peptide can be used to monitor its own aggregation with, therefore, minimal interference on the peptide's native structure, its biochemistry and its spontaneous process of aggregation. Firstly, it is shown that the uorescence of amino acid Tyrosine, naturally present in wild type β-amyloid, responds to the spontaneous aggregation of the peptide. This is achieved by performing time-correlated single-photon counting experiments during the process of the peptide's aggregation into amyloid brils. Through comparison with the well established Thioavin T assay is also demonstrated that Tyrosine decay responds to changes caused by peptide aggregation well before the appearance of the characteristic - sheet structures present in the brils. Then the use of β-amyloid's intrinsic Tyrosine amino acid as a sensor for the pre- brillar stages of aggregation is further tested and researched through a series of experiments with different initial β-amyloid concentrations. Patterns of consistent behaviour are found con rming that Tyrosine can act as a sensor for the formation of oligomers and kinetic information about the oligomerisation process is retrieved. Using this approach a comparison between the oligomerisation kinetics of the two most common variants of β-amyloid is performed. In the process of studying Tyrosine response to the peptide aggregation the accuracy of the discrete exponential decay model used to describe the decays is debated. A model-free analysis is used to study Tyrosine decay photophysics in the β-amyloid peptides throughout the process of aggregation. It is found that Tyrosine decay is a composition of four discrete decay components suggesting the existence of four rotameric forms of the amino acid in the -amyloid peptides. The ndings are further corroborated by molecular dynamic simulations, breaking with the traditional model of three rotameric forms for the uorescent amino acids in protein chains. Finally, the sensing method is used to study the inuence of external uorophores, both associating and covalently bound, on the process of oligomerisation. β-amyloid peptides. The findings are further corroborated by molecular dynamic simulations, breaking with the traditional model of three rotameric forms for the fluorescent amino acids in protein chains. Finally, the sensing method is used to study the inuence of external uorophores, both associating and covalently bound, on the process of oligomerisation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.570606  DOI: Not available
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