Date of Award

Summer 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Lesley H. Greene

Committee Member

Christopher Osgood

Committee Member

Patricia Pleban

Committee Member

Jennifer Poutsma

Abstract

The synuclein proteins α, β and γ which are located in the brain, have been a subject of intense research. Of particular interest is α-synuclein, which is found in misfolded forms in Lewy bodies that are associated with Parkinson's disease. Despite the efforts of researchers across the world, the physiological structure and function of the synucleins remains elusive. In recent years, highly controversial reports by some investigators indicate that in its natural form, α-synuclein exists as a tetramer instead of as an intrinsically unstructured monomer. This dissertation presents results of the experimental and computational analysis of the synucleins. First, we investigated methods of destroying protein fibrils from α-synuclein. We report that low temperature plasma can disrupt synuclein fibrils and proposed that neuronal macrophages can eliminate the resulting structures via phagocytosis. We also conducted inhibition studies to investigate the mechanism by which β-synuclein inhibits α-synuclein fibrillation. Using our experimental conditions, β-synuclein readily formed fibrils while α-synuclein inhibited β-synuclein fibrillation. We show, for the first time, that two fibril forming proteins when incubated together have an inhibitory effect on each other. This important information can be employed in the future development of inhibitors of formation. In order to determine the native structure of the synucleins, we expressed and isolated multimeric forms of the synuclein proteins. We found that expression of β-synuclein in which we did not boil the bacterial lysate. yielded a high molecular weight multimeric β-synuclein form. This study has established the basis for further research with the ultimate aim of forming a well-diffracting crystal that will lead to solving a high resolution X-ray crystallographic structure of the native state. Finally, computational approaches involving bioinformatics and molecular modeling were employed to establish a superfamily for the synucleins. The hypothesis is based on the fact that the structure and function of the synucleins could be inferred from that of their related proteins, whose structure and function have been resolved. Our computational results indicated that the synucleins seem to be orphans in the animal kingdom but share sequence similarity with an endoglucanase enzyme from Acetobacter pomorum bacterium, a CRE-DUR-1 protein from a nematode, a cytochrome c protein from a spiral bacterium and a protein from the Tasmanian Devil. This study led to the development of a proposed evolutionary model for the synucleins, which hypothesized that β-synuclein, encoded by seven exons, is the oldest of the synucleins. α-Synuclein, encoded by 6 exons, evolved to contain amino acid sequences to prevent fibril formation such as the change from threonine 53 to alanine. Together, these results further our understanding of the synuclein proteins from a myriad of experimental and computational vantage points.

DOI

10.25777/h7zm-zk34

ISBN

9781321316414

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