| Two point mutations in the alpha-synuclein gene are genetically linked to autosomal dominant Parkinson's disease. Purified monomeric alpha-synuclein protein is unfolded in solution, but over time a small mol fraction can aggregate to form a heterogeneous mixture of beta-sheet containing soluble oligomers (termed protofibrils) which coexist with the monomeric protein. The monomer/oligomer mixture eventually proceeds to form amyloid fibrils, at which time the majority of the monomer and oligomer is consumed. It is not known which aggregation form of alpha-synuclein is neurotoxic in Parkinson's disease, nor is the pathogenic mechanism known. This thesis demonstrates that a subset of the oligomeric species are capable of permeabilizing negatively charged synthetic phospholipid vesicles via a pore-like mechanism, in vitro. The monomeric and fibrillar forms of the protein have little or no membrane permeabilizing activity, and protofibrils of the Parkinson's disease linked mutants A30P and A53T have a higher specific permeabilizing activity per mol of protein than protofibrils of wild type. Subsequently, a random mutagenesis library of sequence variants was created which codes for approximately 1.5 million different protein sequences (estimated using a new computational method described herein), with an average of two point mutations per sequence. An in vitro aggregation screen of 70 individually expressed and purified variants from the library revealed two double-point mutants which fibrillize significantly more slowly than the wild-type protein. Future examination of the in vivo neurotoxicity of these sequence variants (in a transgenic disease model) may help elucidate the nature of Parkinson's disease pathogenesis. |
