Abstract
The second most common neurodegenerative disease after Alzheimer’s, Parkinson’s disease affects 2% of the world’s population over the age of 65. With no certain diagnostic test or curative treatment, it is imperative to determine the exact pathophysiological mechanisms and proteins behind this disease. It is now known that the aggregates of a 14kDa protein called alpha-synuclein, which manifest themselves in the form of Lewy bodies in doparminergic neurons, are one of the main causes of the debilitating symptoms. The oligomeric forms of alpha-synuclein, particularly the monomer, have been widely studied, but the existence of the tetramer is still subject to much controversy. Although studies now show that the tetramer is the main form of the protein in the body, no study has been able to yield a crystal structure of the tetramer. Based on structural evidence from the multimerization domain of the Nipah virus RNA phosphoprotein, we have carried out site directed mutagenesis that would induce the formation of leucine zippers and therefore stabilize the dynamic structure of wild type alpha-synuclein for crystallization. We have made leucine substitutions at valine residues within the hydrophobic core of the tetramerization domain of GST-tagged wild type alpha-synuclein, to generate two double mutants; V52/55L and V74/77L. When compared to the wild type, the purification and gel electrophoresis of the V52/55L mutant have suggested a change in the physical behavior of the protein and a decrease in the extent of GST tag cleavage by PreScission protease. Future steps would include the optimization of the GST-tag cleavage of the V52/55L mutant, purification of the V74/77L mutant and the generation of other mutants suitable for crystallization.