α-Synuclein Multistate Folding Thermodynamics:  Implications for Protein Misfolding and Aggregation^†

α-Synuclein aggregation has been tightly linked with the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Despite the protein's putative function in presynaptic vesicle regulation, the roles of lipid binding in modulating α-synuclein conformations and the aggregation process remain to be fully understood. This study focuses on a detailed thermodynamic characterization of monomeric α-synuclein folding in the presence of SDS, a well-studied lipid mimetic. Far-UV CD spectroscopy was employed for detection of conformational transitions induced by SDS, temperature, and pH. The data we present here clearly demonstrate the multistate nature of α-synuclein folding, which involves two predominantly α-helical partially folded thermodynamic intermediates that we designate as F (most folded) and I (intermediately folded) states. Likely structures of these α-synuclein conformational states are also discussed. These partially folded forms can exist in the presence of either monomeric or micellar forms of SDS, which suggests that α-synuclein has an intrinsic propensity for adopting multiple α-helical structures even in the absence of micelle or membrane binding, a feature that may have implications for its biological activity and toxicity. Additionally, we discuss the relation between α-synuclein three-state folding and its aggregation, within the context of isothermal titration calorimetry and transmission electron microscopy measurements of SDS-initiated oligomer formation.