The polar protein α-synuclein - An electrifying journey from chaos to order

The accumulation of amyloid fibrils from the protein 𝛼-synuclein is a pathological hallmark of Parkinson´s disease. 𝛼-synuclein is a highly polar protein with a remarkable primary structure that can be divided into three regions: the slightly basic N-terminal region, a central hydrophobic region, and a highly acidic C-terminal tail. The C-terminal tail is of specific focus in this thesis. A change in pH of almost one unit was measured upon fibril formation, indicating upshifts in the pKa values of monomers in fibrils compared to monomers in solution. This can be related to the proximity of the charges in and between different tails surrounding the fibril core. The number of acidic residues significantly altered the rate and pH dependence of 𝛼-synuclein amyloid formation. A decrease in the number of acidic residues within the tail was found to shift the optimal pH range for secondary nucleation to lower pH. These results can be related to a smaller increase in the pKa values upon fibril formation for the mutants in comparison to the wild-type, resulting in a downshift in the pI value and thus affecting the relative contribution of various intra- and inter-molecular interactions. Furthermore, the interaction of free monomers with the fibril surface of two chemically identical but structurally distinct morphologies was studied. The two morphologies formed under identical conditions, presumably due to similar kinetic barriers. In both cases, the positively charged N-terminus was found to be attracted to the negatively charged fibril surface, to what extent differed between the two morphologies. These results further emphasize the role of electrostatic interactions in the amyloid formation, with particular relevance for secondary nucleation. The solubility and, thus, the stability varied between the two fibril morphologies. However, with time, the samples will become dominated by the more stable fibril morphology. The aggregation of 𝛼-synuclein in the presence of the molecular chaperone DNAJB6b was studied under mildly acidic conditions, showing a retardation effect at sub-stoichiometric ratios. The results show strong indications of the formation of 𝛼-synuclein-DNAJB6b co-aggregates. The co-aggregates were found to have higher apparent solubility, which may be related to the high chemical potential of the chaperone alone. The free energy of the system as a whole may thus be lower for co-aggregates compared to 𝛼-synuclein fibrils co-existing with free chaperone in solution. Lastly, the method "photo-induced cross-linking of unmodified proteins" was optimized for studying 𝛼-synuclein oligomers. We find that good control of the reaction time is crucial.