Synuclein plasticity: the Achilles’ heel of nerve function linked to the onset of Parkinson’s disease

Abstract

Lewy bodies – the hallmarks of Parkinson’s disease – are majorly constituted of aggregates of the presynaptic protein alpha-synuclein. The molecular mechanism of alpha-synuclein aggregation through which it changes dramatically from a soluble disordered monomer to insoluble structured fibrils remains unknown. As an intrinsically disordered protein, alpha-synuclein does not have a specific three-dimensional structure, but rather behaves mostly as a meta-stable ensemble of highly dynamic conformers, and as such undergoes rapid kinetics, making it almost impossible to measure its conformational changes with most techniques. Millisecond amide hydrogen exchange can provide valuable insights on the dynamic behaviour of proteins, especially at flexible regions. Thus, the work in this thesis reports on the development of methods and tools for hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and the application of these for the study of aSyn under physiological conditions. In the first part of this thesis, high resolution on the alpha-synuclein monomer was achieved over two dimensions: time and space. Using a novel in-house rapid- mixing quench-flow instrument, hydrogen/deuterium-exchange mass spectrometry data on alpha-synuclein on the millisecond timescale was attained. Furthermore, using a ‘soft’ gas-phase mass spectrometry fragmentation technique called Electron Transfer Dissociation, structural resolution in the protein increased. The second part of this work focuses on the development of a software, HDfleX, in an effort to primarily automate the HDX-MS workflow and allow the merging of HDX-MS data at different levels: bottom-up, middle-down and top-down. The rest of the thesis uses the tools and methods developed earlier on to explore the effects of different solution conditions (cellular compartments and salt cations) on the monomeric conformations of aSyn, and how these correlate to the different stages of aggregation and the ensuing fibril polymorphs. Altogether, the achievements in this work will allow us to better understand the plasticity of the alpha-synuclein monomer as it cycles through different local environments.