Abstract by Ersoy Cholak
Structural and biophysical characterization of α-synuclein:lipid co-structures
Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting millions of lives worldwide, with no cure currently available. A major hallmark of PD is the deposition of lipid-rich protein inclusion bodies – termed Lewy Bodies (LB) – within neurons of the brain. These LBs contain a high proportion of the presynaptic protein, α-synuclein (aSN) in aggregated fibril forms. aSN is a small (140 residues in human), intrinsically disordered protein but it adopts a partially α-helical structure upon binding to membrane bilayers. Although the biological function of aSN remains unclear, its structural adaptation is believed to be associated with trafficking and/or fusion of synaptic vesicles as well as neurotransmitter release. Ultimately, outlining the interaction between aSN and membranes plays a key role for understanding the underlying biology in both physiological and pathological conditions. Studying them at the molecular level is, however, hampered by heterogeneity within the protein population, which is believed to cover an ensemble of various free and membrane-binding conformations in the cell.
This thesis presents experimental biophysical studies addressing the interaction between monomeric aSN and model lipid membranes, specifically, small unilamellar vesicles (SUVs) of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG). Structural and biophysical characterization of the isolated membrane-bound form of aSN and aSN:lipid co-structures was performed using size exclusion chromatography, circular dichroism, nuclear magnetic resonance spectroscopy, X-ray diffraction and small angle X-ray scattering.
The results presented here reveal that the formation of aSN:lipid co-structures (i.e., POPG SUVs decorated with aSN), is highly dependent on avidity between the N-terminal tail (residues 1-14) of the protein anchoring into the hydrophilic outer leaflet of the bilayer and interaction of its membrane associated fragment (residues 15-100) with the lipid the head groups. Additionally, N-terminal acetylation (NTA) does not change the overall mechanism of aSN:lipid co-structure formation. Once this avidity is broken, aSN membrane affinity is compromised and co-structure formation suppressed. Formation rate of the co-structures is affected by several factors such as NTA, lipid/protein molar ratio and protein concentration. Furthermore, I also show that aSN:lipid co-structures escalate the fibrillation process of monomeric aSN by enhancing nucleation compared to pure POPG SUVs.
These findings represent a step towards a better understanding of aSN membrane interaction, as well as protein function and dysfunction in the brain.