Nikhil Virani, Meghan Mattheny
Faculty Sponsor: Jeffery Myers
Almost one million Americans suffer from the devastating effects of Parkinson’s Disease, making it the second most common neurodegenerative disorder behind Alzheimer’s Disease.1A vital action in the fight against neurodegenerative disorders such as Parkinson’s Disease is understanding the mechanisms by which normally folded proteins develop pathological properties. The α-synuclein fibrils (cylindrical conglomerates of protein) called Lewy Bodies or Lewy Neurites are a known characteristic of Parkinson’s, but the cause and progression of this misfolding and aggregation is still under investigation. Our ongoing research seeks to investigate the complex mechanisms of fibrillation utilizing analysis by mass spectrometry. The production and purification of aS from E.coli can provide a useful model when investigating aS behavior in the human brain. We utilized bacterial transformation using a plasmid to promote the production of the human protein aS in the bacteria. Next, bacterial optimization was carried out to maximize production of aS and fitness of the chosen colonies. To separate the protein from the other cellular debris, we used centrifugation, dialysis, fast protein liquid chromatography, and gel filtration chromatography. After we created two samples of aS from different colonies, we proceeded to perform different tests on the purified protein. One experiment run was the addition of pepsin to the protein solution. The aim was to determine pepsin cleavage sites. Resulting samples, taken every two hours after introduction of the enzyme, run on an SDS page cell showed the acidic conditions required by the pepsin cleaved the protein almost completely in half. As time progressed more fragmentation was observed. Future examination on Mass Spec. could elucidate more specifically the composition of the fragments. The second experiment was constructing the fibrils using fluorescence to measure the rate of fibrillation and measure the hydrogen exchange rate of aS to determine its characteristics. Thioflavin was used with the samples to measure the rate at which aS was fibrillated. Hydrogen exchange was performed using Deuterium Oxide to replace the hydrogens in aS to deuterium, an isotope of hydrogen and used Mass spectrometry to learn more about protein conformation and dynamics.
