Untangling Fibrillogenesis: Investigations of the Mechanisms of Amyloid Formation

  • Benjamin Gardner

    Student thesis: Phd


    A number of diseases share a common mechanism, which is the misfolding of proteins leading to theformation of insoluble fibrils through a process termed fibrillogenesis. Diseases where fibrillogenesis is central to the pathology include CJD, Huntington's disease and Alzheimer's disease, all three beingirreversible fatal neurodegenerative disorders. Therefore, an improved understanding of the mechanistic process involved is crucial if there are to be developments in the treatment for these diseases. However, the conventional approaches routinely used in the structural analysis of biomolecules such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopies are restricted in their application to dynamic systems; such as for unfolded proteins, or in studying rapid changes in structure, both of these situations are usually observed in protein misfolding and fibrillogenesis. Vibrational spectroscopic techniques including Raman spectroscopy, Raman optical activity (ROA), tip enhanced Raman spectroscopy (TERS) and deep UV resonance Raman spectroscopy (DUVRR) have been shown to be particularly well suited for the study of biomolecules. This has been found to be especially so for monitoring structural transitions and protein dynamics. Therefore, these techniques are being increasingly used in the study of fibrillogenesis.The work in this thesis focuses on the use of vibrational spectroscopies to investigate the mechanisms of fibrillogenesis for bovine insulin and lysozyme. Through monitoring the fibrillogenesis of bovine insulin in real time with Raman spectroscopy, it was possible to readily identify distinct phases in the transitions from the native monomeric state to β-sheet rich fibrils. This was possible through combining different analytical methods including: full width half height analysis (FWHH), 2D correlation approaches and principal component analysis (PCA). It was also shown that the pH of the sample, while not appearing to affect the initial structure, had a significant effect on the rate of fibril formation. Moreover, temperature quenching of the sample when in the β-sheet intermediate was shown to disrupt fibril formation and allow disassociation and recovery of the initial native state.The structure of the independent A and B chains of insulin was shown to be sensitive to pH, which also had a significant effect on fibrillogenesis mechanism. This was especially so for the B chain of insulin where at pH 3 full fibrils were formed, while at the extreme of pH 1 a mixture of spherulites and fragmented fibrils formed. Moreover, using TERS it was shown that there were significant differences in the surface chemistry of the A and B chain fibrils with respect to secondary structure composition.Raman spectroscopy was used to show that different Hofmeister ions including Na+ and Ca2+ affect the initial unfolding of lysozyme differently, indicating different mechanisms of interaction. Further to this, ion concentration had a dramatic effect on the stability of the partially unfolded intermediate, whereby increases in salt concentration rapidly increased the rate of β-sheet formation.The effect of the protonation state on the structure of the 20 common amino acids was also investigated with Raman and ROA spectroscopy. This study extended to the use of density functional theory (DFT) calculations on Val, which confirmed that changes in pH have an extensive influence on the structure and dynamics of amino acids. The changes were shown to beyond the simple addition of a proton, with variations in side chain environment and hydrogen bonding also being observed.In order to facilitate spectral treatment and post-acquisition analysis of the large number of Raman and ROA spectra collected in this project, a graphical user interface (GUI) toolbox was developed for use within Matlab. This toolbox contained several common data treatment pre-requisites which are crucial for reliable post-acquisition analysis.Overall, this work
    Date of Award31 Dec 2014
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorEwan Blanch (Supervisor) & Jonathan Waltho (Supervisor)

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