Elucidating the Structural Dynamics of Alpha-Synuclein by Structural Mass Spectrometry

Parkinson’s disease (PD) is characterised by the deposition of insoluble Lewy Bodies (LBs) in dopaminergic neurons in the brain. LBs are primarily composed of a-Synuclein (aS), a 140-residue, intrinsically disordered protein which can self-associate and undergo a transition from disordered monomers into ordered b-sheet rich amyloid fibril architectures. Characterising the structural properties of early intermediates in aS amyloid assembly is crucial towards elucidating amyloid assembly mechanisms. This thesis presents the development and application of structural mass spectrometry (MS) based techniques to study the structure and dynamics of N-terminally acetylated aS (aSNTA). Findings capture how the conformation of aS correlates with its amyloid propensity. The conformational ensemble of aS along with variants which decrease/abolish amyloid assembly is shown. Ion mobility MS shows that monomeric aSNTA exists as a conformational ensemble populating partially compact conformational families in equilibrium with extended conformational families and this thesis explores the effect of perturbing the aS conformational ensemble on its amyloid assembly kinetics. aS is negatively charged under physiological conditions and is known to bind divalent metal ions. Upon addition of Ca2+, Mn2+ or Zn2+, multiple binding events occur and the conformational ensemble of aSNTA is shifted to compact conformations and the rate of amyloid assembly is increased. Oligomeric species populated during aS amyloid assembly are considered toxic drivers of neurodegeneration in PD but are difficult to study due to their transient form and heterogeneity. In this thesis, hydrogen-deuterium exchange MS is used to characterise the role of the N-terminal region in trapped oligomer assembly. Overall, this thesis aims to shine light on the use of structural MS to investigate the structure and dynamics of transient, heterogeneous proteinaceous species. Evidence is provided for the accelerating role of aS compaction in amyloid propensity and provides a foundation for the further development of structural MS based methodologies.