Affinity of Alpha-Synuclein and Lipid Vesicle Interaction

Sammanfattning: Alpha-synuclein is a protein involved in several neurodegenerative diseases, such as Parkinson’s, where it has been observed to form amyloid fibrils or Lewy bodies. However, how or to what extent alpha-synuclein is involved is not known. I have in this thesis been conducting experiments in order to design an experimental model system to study how alpha-synuclein interacts with a cell membrane. This system consists of giant unilamellar vesicles (GUVs) made up of various lipid species, including 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). The vesicles are loaded with a fluorescent dye (fluorescein) which starts to leak out if the membrane becomes permeable. It was observed that GUVs with a diameter exceeding 20 μm could be made of different lipids and filled with fluorescein. The GUVs were photobleached and the movement of fluorescein in and out of them was measured. Upon addition of alpha-synuclein it was observed it caused no detectable structural damage of the GUV membrane during 20 minutes. In summary, I have investigated the steps in setting up a general method of studying protein-lipid interactions, where the most ideal way to monitor GUV membrane permeability was found, with specific parameters regarding lipid concentrations to ensure membrane stability.