α-Synuclein Binding Copper (II) in the Presence of Lipid Bilayers

Parkinson’s disease (PD) is the most prevalent age-related motor disorder, affecting ~1.8% of individuals over the age of 65. Parkinson’s disease involves the degeneration of dopaminergic neurons of the substantia nigra pars compacta, and the presence of pathognomonic cytosolic inclusions in surviving neurons, known as Lewy bodies (LB). Alpha-synuclein (α-syn), an intrinsically unstructured protein, is found in high concentration in neurons, localized mostly at presynaptic terminals, and is the major component of LB.  Epidemiological studies have shown that long-term exposure to heavy, polyvalent metals, such as Cu²⁺, increase the risk of PD mortality. An interaction between Cu²⁺ and α-syn has been identified, whereby α-syn strongly binds 1 equivalent of Cu²⁺ and coordinates at the N-terminal amine, Asp2 amide backbone and side chain carboxyl group, and the His50 imidazole. α-Syn is known to associate with lipid membranes, where it is largely in α-helical form. α-Syn in lipid-bound form appears to be incompatible with simultaneous Cu²⁺ coordination by the N-terminus and His50. Our interest is to characterize the association between α-syn and Cu²⁺ in the presence of lipid membranes. To examine the effect of copper binding of the Asp2 side-chain residue in the presence of lipids, an Asp2 to Ala mutation (D2A) will be made by site directed mutagenesis of wt α-syn. Circular dichroism spectroscopy will be used to determine the secondary structure of D2A. The stoichiometry and affinity of the D2A mutant binding Cu²⁺ when lipid-bound will be characterized by electron paramagnetic resonance (EPR) titrations, and an EPR competition technique, respectively.