Isolation of Intricate α‐Synuclein Assemblies Across the Dynamic Landscape

Abstract

Cardinal features of Parkinson’s disease (PD) include the development of granular inclusions known as Lewy bodies that are enriched with aggregates of α‐synuclein (αS). αS has long been established as an intrinsically disordered protein, and the array of its physiologically relevant oligomeric conformations is ever‐expanding. In recent years, a higher order conformation was identified as a helical tetramer that is tailored by buried hydrophobic interactions and is distinctively aggregation resistant. The monomer and tetramer reside in a dynamic coexistence within cells, yet the canonical mechanism by which the tetramer assembles remains elusive due to the various folding patterns and low energy barriers involved in αS structural transitions. A shift from metastable tetramers to the monomeric form likely serves as a mechanism for disease initiation. The underlying pathology causing this shift remains unknown, but the importance of understanding tetramer stability and disassembly has therapeutic potential that cannot be overemphasized. Hence, a platform for investigating analytical approaches to stabilize tetrameric αS is warranted, as are methods to discriminate αS conformational states. Isolation of tetrameric αS is complicated by its dynamic nature, thus thorough bioanalytical studies on this conformer have been hampered by accessibility issues. We recently developed a robust recombinant expression platform that enables purification of native tetrameric αS without any detergents, crosslinkers, or other structure‐modifying additives by the sequential combination of anion exchange and size exclusion chromatography steps. The successful isolation of this intricate αS conformer is confirmed through various analytical methods, including advanced light scattering techniques, mass spectrometry, immunoblotting, and circular dichroism spectroscopy for secondary structural analyses. The development of multifaceted and innovative approaches that are necessary to tease apart the dynamic landscape of αS quaternary structure will be presented. Moreover, novel methodology to facilitate localized surface plasmon resonance analysis of this intricate conformer will be highlighted as well as the effects of PD‐relevant structural modifiers present in cellular milieu, including metals and/or liposomes, strengthening the application of this platform to advance tetrameric αS as a therapeutic target. The talk will conclude with new insights on strategies to shift the conformational equilibrium towards tetramer in mixed populations as a means to thwart αS misfolding and aggregation.Support or Funding InformationSupported by NIH R01 GM134015, NCATS UL1TR002649 & CCTR Endowment Fund, VCoA ARDRAF Award No. 18‐7, and NIA L‐13046.