The structure of the native α-synuclein ensemble determined using a combination of structural proteomics and discrete molecular dynamics simulations




Brodie, Nicholas I.

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In Parkinson’s disease and other Lewy Body disorders, aggregation of the protein α-synuclein results in the degeneration of nervous tissue. Under normal conditions, the α-synuclein protein is abundant in neurons, where it assists in the formation of vesicles and the reuptake of neurotransmitters. However, under some conditions the protein will undergo a prion-like misfolding conversion and ultimately be converted into a fibrillar form, which makes up the bulk of the protein content of Lewy bodies. Currently, our understanding of the initial structural changes involved in the conversion of this protein into a toxic oligomeric form is hindered by the limited availability of structural data on the native, intrinsically disordered protein. Helping to define a structural ensemble for this protein would be a first step towards the development of a model for the misfolding and oligomerization process of this protein. The research hypothesis for this dissertation is that the α-synuclein protein adopts a conformational ensemble of structures which can be elucidated using structural proteomics, and that some of these conformations have features which may lead to an increased propensity to form oligomers. In order to test this hypothesis, I utilized a variety of structural proteomics tools. These included chemical crosslinking for the discovery of distance constraints which can be used for molecular modelling, surface modification experiments which determine the propensity for particular residues to reside on the protein surface, hydrogen-deuterium exchange measurements for determining the presence or absence of secondary structure, and molecular modelling, which will be performed by collaborators at the University of North Carolina. In order to help answer these difficult structural questions, I developed a variety of new structural proteomics techniques including photo-reactive, non-specific crosslinking reagents, ultraviolet photo-dissociation for protein fragmentation during hydrogen deuterium exchange experiments, and, most importantly, in collaboration with the University of North Carolina, I developed a computational pipeline for determining protein structures by directly incorporating distance constraints into discrete molecular dynamics simulations. These new techniques were first tested on several model proteins in order to verify their effectiveness, and were then used in combination with already-established structural proteomics techniques to model new ensembles for the native synuclein protein. This ensemble structure indicates that in vitro the synuclein protein adopts an ensemble of 4 distinct structures, each with some transient secondary structure. In particular, the most populated structures in the ensembles possessed secondary structure motifs in regions known to be important for oligomerization, and stabilization of these transient structures is likely to be a key component of the conversion to the oligomeric form of the protein.



Synuclein, Discrete Molecular Dynamics, Crosslinking, Structural Proteomics, Surface Modification, Intrinsically Disordered Protein, Protein Modelling, Protein Crosslinking, Photoreactive Protein Crosslinkers