Utilising fluorescence microscopy to visualise the dynamics and interactions of molecular chaperones and α-Synuclein.

In Parkinson’s Disease (PD), the protein α-synuclein and its early stage oligomers have been implicated as the primary cause of neuronal toxicity. Molecular chaperones play an important role in maintaining protein homeostasis in such diseases. The dynamic nature of the chaperone cycle and its interaction with the α-synuclein substrate makes it difficult to characterise by traditional biochemical techniques. Using a range of fluorescence microscopy approaches I show that α-synuclein exists in a concentration dependant dynamic equilibrium between monomeric and multimeric states, with rapid exchange of subunits between species. Dilution leads to rapid monomerisation and loss of structure followed by a slow recovery of higher order species. Chaperones from the Hsp40/Hsp70 machinery interact preferentially with the dissociated monomeric α-synuclein and delay the reassociation of subunits to higher order species implicating the multimer form as the potentially unknown native state of α-synuclein. Using sinlge-molecule techniques I also studied the membrane binding properties of the larger α-synuclein oligomers associated with toxicity. This method allowed the visualisation in real-time of the perforation of lipid vesicles in the presence of α-synuclein oligomers and characterisation of the ability of chaperones to prevent this process. A greater understanding of the mechanism of chaperone-mediated modulation of disease proteins and their preferential interactions will provide insights into the early stages of the progression of Parkinson’s disease