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Biophysical approaches to characterising protein-protein interactions and intermediate species in protein aggregation 10.17863/CAM.65349 Xu, Catherine Knowles, Tuomas Dobson, Christopher The phenomenon of protein misfolding and aggregation has been associated with over 50 human diseases, including Parkinson’s disease (PD). While the hallmark deposits in aggregation-associated diseases are primarily composed of fibrillar species, intermediate oligomeric species that form during the aggregation process are believed to be a major cause of toxicity. Such species are relatively poorly characterised due to several challenges that render them inaccessible to most conventional techniques; oligomers are only present at extremely low concentrations in the aggregation reaction, and are additionally highly heterogeneous and often transient in nature. In this thesis, I present complementary approaches to address these difficulties. Firstly, an ensemble of stable, kinetically trapped oligomers with varying biophysical characteristics was established, enabling detailed structure-toxicity relationships to be investigated. Secondly, a method for the simultaneous single-molecule level characterisation and fractionation of oligomeric species under native conditions was established and applied to studying intermediate species in Parkinson’s disease-associated protein aggregation. Finally, I present a general approach to optimising experimental design, which maximises both the efficiency and information gain of experiments, and demonstrate its validation and application to several experimental systems. University of Cambridge 2020-09-29 Thesis Doctoral Doctor of Philosophy (PhD) eng Herchel Smith Fund https://www.repository.cam.ac.uk/handle/1810/318229 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f2829d23-f685-4475-a4cf-ae167efd5f37/download 73d815a7b726419d78c4379d1574c5e7 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/14d3dc54-efd7-4541-be62-5d4d0dd8a420/download 353adac0d1ebdfd65ab16480263c3c87 https://www.rioxx.net/licenses/all-rights-reserved/ Protein aggregation Amyloid Oligomers Alpha-synuclein Microfluidics