Probing surface-mediated protein aggregation via FRET-induced lifetime changes

Alpha-synuclein (aSyn) aggregation in the brain is a distinctive pathological feature of Parkinson's disease (PD). Much remains unknown about the molecular details of aSyn aggregation, namely the mechanism for its onset and propagation, and how this process leads to neuron dysfunction and ultimately death. A substantial fraction of aSyn molecules in neurons are bound to phospholipid membranes, where binding affinity is dependent on membrane polarity and curvature. Membrane-induced aSyn aggregation is hypothesized to be a pathologically relevant event in PD. Notably, lipid membrane surfaces may act as a platform upon which nucleation can occur. Surface-mediated protein aggregation can be studied via fluorescence resonance energy transfer (FRET) interactions between fluorescently labeled membrane surfaces and proteins, where the donor fluorescence lifetime is indicative of the degree of proximity. We describe progress on the detection and understanding of surface-mediated aSyn aggregation on artificial membranes made from anionic phospholipids. This is achieved through FRET-induced fluorescence lifetime changes recorded by a custom-built fluorescence lifetime imaging microscope. Additionally, the affinity of aSyn to various membranes in SY5Y neurons is explored.