Pulling apart the intermolecular interactions of the Parkinson’s disease linked protein alpha synuclein

Amyloidoses are a group of protein misfolding diseases that are characterised by the abnormal accumulation of highly ordered filamentous assemblies known as amyloid. This phenomenon is associated with more than 50 human diseases, some of which are the most debilitating disorders that threaten human health today. Many of these disorders have age as the main contributing risk factor and, therefore, pose an ever-increasing risk in the developed world with aging societies. Despite intense research, much remains unknown about the fundamental processes driving protein aggregation in these diseases and there are few disease modifying treatments available. A protein that undergoes amyloid formation and causes disease is the intrinsically disordered neuronal protein α-synuclein (αSyn), the aggregation of which leads to several diseases including Parkinson’s disease (PD) which is the second-most common neurodegenerative disorder that affects 2–3% of the population ≥65 years of age. Importantly, the toxic species on the aggregation pathway are difficult to identify and determine in molecular detail. This thesis was motivated by this fact and aimed to study the initial intermolecular events in αSyn self-assembly (dimerisation) on a single molecule scale. Single molecule force spectroscopy (SMFS) methodologies were therefore utilised in order to study these early protein-protein interaction events. A display system was firstly designed and validated in which small regions of highly aggregation-prone sequences can be presented in a protein scaffold in a robust and reproducible manner for SMFS studies. It was demonstrated that intermolecular interactions of these sequences could be analysed by implementing this system. A novel heterodimeric interaction between the central aggregation-prone regions of αSyn (residues 71-82) and the same region of its human homologue γSynuclein (γSyn), were revealed by using this system. Further study led to the finding that this interaction played a role in the inhibiting the aggregation of αSyn. The dimerisation interaction of full length αSyn has also been analysed in this thesis and several important findings have been demonstrated. The SMFS experiments show that force-resistant structure forms in the dimeric species of αSyn and that this structure is dependent on the environmental conditions. SMFS utilising different immobilisation regimes of αSyn have also allowed the location of a novel interaction interface involving the N-terminal region of the protein. Further SMFS experiments investigating the effects of salt and hydrophobicity have on dimerisation, alongside bioinformatics analyses of the protein sequence led to the hypotheses that the dimeric interaction is driven by hydrophobic stretches in the N-terminal region, but modulated by local electrostatics. In vitro aggregation assays and SMFS on non-aggregation-prone synuclein homologues (β- and γSyn) indicated that that this interaction is protective against aggregation, considering these finding with existing literature prompted speculation that the interactions observed in SMFS may indeed be physiologically relevant. This may therefore be an important finding in regards to targeting the aggregation process with disease modifying agents.