Towards an understanding of amyloidogenesis: a structural and biophysical analysis of transthyretin

Yee, Ai Woon

Abstract

Human transthyretin (TTR) is heavily implicated in a range of fatal amyloid diseases. The propensity to form amyloid varies between wild-type TTR and various mutants. In all cases this occurs by tetramer dissociation to a monomeric form, which in turn polymerises into intermediates that ultimately assemble into amyloid fibrils. Numerous X-ray crystal structures of TTR mutants have been determined over the last 40 years. These structures have all turned out to be very similar, shedding little light on the widely varying amyloid-forming properties. The goal of this PhD project was to investigate the neutron crystal structures of these mutants – on the basis that factors that are difficult to identify using X-ray crystallography (protonation states, hydrogen bonding, hydration) may be implicated in the varying TTR stabilities. High-resolution neutron crystallographic studies of two TTR mutants were carried out. These mutants were chosen for their strongly contrasting behaviour. The S52P mutant is highly amyloidogenic, whereas the T119M mutant is resistant to amyloid formation. As part of these studies, a unique way of measuring and exploiting H2O/D2O back-exchange was uncovered – allowing the stability of specific regions in the S52P structure to be probed. A wide range of mass spectrometry measurements were also carried out. These initially focused on the assessment of the impact of deuteration of the proteins produced for crystallographic work. However, in addition, the impact of different mutations on the subunit exchange kinetics of TTR tetramers was also studied. Finally, the impact of binding of a small-molecule ligand, tafamidis, to the S52P mutant was investigated through a combination of structural and biophysical studies. Collectively, these results have been placed in the context of the overall knowledge of TTR amyloidosis, and an exciting perspective for work is given.

Citation

Yee, A. W. (2018). Towards an understanding of amyloidogenesis: a structural and biophysical analysis of transthyretin. (Thesis). Keele University

• Thesis Type: Thesis
• Additional Information: Digital copy available upon request from the Archives https://forms.office.com/e/sRWE7eQWgU - third party copyright content preventing thesis being published online. For access to the hard copy thesis, check the University Library catalogue.
• Award Date: 2018-03