Experimental and computational analysis of the synucleins

EXPERIMENTAL AND COMPUTATIONAL ANLALYSIS OF THE SYNUCLEINS PROTEINS Sr. Agatha Munyanyi Old Dominion University Director: Dr. Lesley Greene The synuclein proteins a. (3 and y which are located in the brain, have been a subject o f intense research. O f particular interest is a-synuclein, which is found in misfolded forms in Lewy bodies that are associated with Parkinson’s disease. Despite the efforts o f researchers across the w orld, the physiological structure and function o f the synucleins remains elusive. In recent years, highly controversial reports by some investigators indicate that in its natural form, a-synuclein exists as a tetramer instead o f as an intrinsically unstructured monomer. This dissertation presents results o f the experimental and computational analysis o f the synucleins. First, we investigated methods o f destroying protein fibrils from a-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 p-synuclein inhibits a-synuclein fibrillation. Using our experimental conditions, |3-synuclein readily formed fibrils while a-synuclein inhibited p-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 o f inhibitors o f formation. In order to determine the native structure o f the synucleins, we expressed and isolated multimeric forms o f the synuclein proteins. We found that expression o f P-synuclein in which we did not boil the bacterial lysate, yielded a high molecular weight multimeric Psynuclein form. This study has established the basis for further research with the ultimate aim o f forming a well-diffracting crystal that w ill lead to solving a high resolution X-ray crystallographic structure o f 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 o f the synucleins could be inferred from that o f 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 w ith an endoglucanase enzyme from Acetobacter pom orum 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 o f a proposed evolutionary model for the synucleins, which hypothesized that P-synuclein, encoded by seven exons, is the oldest o f the synucleins. a-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 o f the synuclein proteins from a myriad of experimental and computational vantage points.