Development of Biophysical and Chemical Methods for Investigations into the Polymorphic Nature of Amyloid Fibril Structures

Protein misfolding and self-assembly into the amyloid state is associated with a range of neurodegenerative diseases. These diseases affect an increasing number of patients every year and have significant detrimental human and economic impacts. Despite sharing the cross-β core architecture, amyloid fibrils exhibit polymorphism, which is likely to underpin the relationship between structure, function, and dysfunction. In this work, the nature of amyloid fibril structural polymorphism is investigated using chemical conjugation and biophysical methods. Amyloid-DNA conjugates were made, with the aim of altering amyloid suprastructure and thus modulate the transmission of amyloid between cells, as well as develop modular nanomaterials. A modified variant of the Sup35 prion protein was used as a model system to which complementary DNA strands or a nucleobase analogue were conjugated via thiol-maleimide reactions. Atomic force microscopy, a nanoscale imaging technique, was used to characterise the modified Sup35 fibrils. Furthermore, AFM image analysis methodologies were advanced through the development of image deconvolution and 3D reconstruction algorithms. This approach led to both in silico correction of an imaging artefact and an approximate doubling of local resolution. The AFM analysis approach was further improved by integrating data from cryo-EM with AFM 3D envelopes, in a developed comparative morphometrics approach. This was demonstrated on dGAE tau amyloid and cryo-EM density maps of tau fibrils from various tauopathies, as well as heparin-induced in vitro formed fibrils, showing that in vitro self-assembled dGAE is a physiologically relevant model system as it is similar to PHF fibrils from Alzheimer's disease patient brain tissue. Finally, a systematic meta- analysis of structural features across all amyloid fibril atomic models in the PDB and EMDB was carried out to investigate the structural basis of the different biological effects of amyloid. The work in thesis contributes to improving our fundamental understanding of amyloid structure-function relationships.