Development of mass spectrometry approaches to characterize intrinsically disordered proteins α-synuclein

Subtitle:a key protein in Parkinson’s disease

Over the last years the use of mass spectrometry (MS) in the structural biology field has significantly increased. Native MS approaches, structure-sensitive digestion and fragmentation, crosslinking and labeling techniques coupled to MS gained their position in the structural MS field. The information obtained includes the protein mass, subunit stoichiometry of protein complexes, which protein regions are solvent exposed or buried inside the structure, ligands that interact with the protein, ligand stoichiometry, ligand binding sites, general shape and conformational changes of the protein, protein-protein interaction sites and of course the protein sequence with eventual mutations or post translational modifications (PTMs). An important class of proteins are intrinsically disordered proteins (IDPs), that account for over 30% of all eukaryotic proteins. With a (partial) natively disordered structure these proteins are very challenging to characterize, due to their dynamic and heterogeneous conformational ensemble. Development and use of structural MS is important to further elucidate IDP structure since MS methods can cope with their flexible and dynamic nature. In this thesis the IDP alpha synuclein (α-syn) is investigated using various MS approaches, to further characterize this protein and show the possibilities of MS as a structural technique in the challenging field of IDP characterisation. α-syn consists of 140 amino acids, is mainly expressed in presynaptic nerve terminals and plays a major role in the development of Parkinson’s disease (PD). α-syn monomers can aggregate and form intermediate structures such as oligomers, which then further aggregate and form mature α-syn fibrils. Various factors, e.g. mutations, PTMs, ligands, pH and the presence of biological membranes, can affect this aggregation pathway. It is important to know how these changes occur at the molecular level to gain more understanding about aggregate formation and how this might be tackled. Using native and ion mobility (IM) MS it was investigated how far we can characterize interactions and conformational changes of α-syn monomers, and we show how instrumental advances in MS contribute to the structural IDP field. Native IM-MS was also used to determine possible structural effects of disease related mutations and relevant PTMs of α-syn. Finally we show that MS-based techniques can bridge the gap between molecular events that determine monomer conformations and the resulting aggregate structures. In general the value, relevance and importance of using MS-based techniques in the structural biology field to study challenging systems such as IDPs to characterize their full conformational ensemble and aggregation pathway is highlighted.

Publication year:2021

Keywords:Doctoral thesis

Accessibility:Open