In vitro fibrillization of proteins into amyloid fibrils provides critical insights into the factors influencing protein aggregation and has a key role in understanding the molecular basis of several neurodegenerative diseases caused by amyloids. α-synuclein (αSyn), an intrinsically disordered protein implicated in Parkinson’s disease, fibrillizes in vitro with ease and is shown to exhibit appreciable structural polymorphism. This inherent polymorphism is a major obstacle in elucidating structural features and understanding the fibrillization process. This thesis explores the fibrillization characteristics of αSyn and the use of αSyn fibrils as scaffold for creating hybrid bionanostructures. We show that fibrillization of αSyn can be carefully modulated by solution conditions to produce morphologically homogeneous fibrils of wt and the disease mutants at the plateau phase of the thioflavin-T assay. Interestingly, all of the αSyn sequences form homogeneous fibrils albeit of distinct morphologies, visualized using atomic force microscopy. The same homogeneous fibril samples, however, show considerable polymorphism in the early phase of fibrillization. Moreover the wt fibrils mature over a period of six months while the disease mutants form stable fibrils. In addition to studying the longitudinal morphological changes in αSyn fibrils, the ability to produce homogeneous fibril population provided us with a singular opportunity to investigate the polymorph specific features of αSyn fibrillization. Subsequent comparative studies on two different morphologies revealed that distinct morphologies present typical binding sites for thioflavin-T molecules, resulting in type-specific fluorescence behavior in the thioflavin-T assay. Furthermore, heterologous seeded aggregations between these morphologies demonstrated that conformational compatibility between the seed and the soluble monomer is important for seed elongation. We also explored the functionalization of cysteine carrying αSyn (A140C) with gold nanoparticles for producing conductive nanoscale assemblies. Overall we show that αSyn fibrils with different morphologies have distinctive biochemical and biophysical properties, and are a promising scaffold for functionalization.
|Award date||3 Feb 2016|
|Place of Publication||Enschede|
|Publication status||Published - 3 Feb 2016|