Power of kinetic growth curve analysis in determining the mechanism of amyloid fibril formation
Gillam, Jay Ellen
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Misfolding and accumulation of insoluble protein aggregates in the form of amyloid fibrils is associated with a number of prevalent and debilitating mammalian disorders. In addition, amyloid-like nanostructures exhibit robust material properties, biological compatibility and replicative properties, making them of particular interest in the development of novel nanomaterials. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. The amyloid hypothesis is that since amyloid-like fibrils share a common core structure, they also share common formation mechanisms. Utilising a combination of turbidity and extrinsic fluorescence techniques this thesis provides insight into the diagnostic strength of simple, inexpensive kinetic measurements of aggregate growth. These simple techniques are found to be capable of delivering a substantial amount of information about the growth mechanisms controlling aggregation, and the effect of solution and environmental conditions, forming a solid basis for further investigation. Two competing fibrillar pathways are observed for hen egg white lysozyme at low pH in the presence of salt. These two pathways, leading to the formation of either curvilinear, worm-like fibrils or to the more widely recognised rigid, straight fibrils are not particular to hen egg white lysozyme, and similar competition may affect growth curve analysis in many other protein assays, including a-synuclein. Many proteins aggregate in the presence of membranes and detergents, and the kinetics of a-synuclein aggregation in the presence of SDS are strongly influenced by SDS concentration. Most descriptions of amyloid fibril growth currently lack heterogeneous nucleation events, and these may be important for predicting aggregation of membrane-active species in vivo. It is clear that simple analytical solutions to growth models are unable in many cases to capture the complexities of filament growth. Even in relatively simple in vitro experiments different growth processes can dominate growth rate over time, competing fibrillar species can result in composite kinetic growth signals and some growth mechanisms have not yet been sufficiently incorporated into an overall description of fibril growth.