Comprehensive Structural and Thermodynamic Analysis of Prefibrillar WT α-Synuclein and Its G51D, E46K, and A53T Mutants by a Combination of Small-Angle X-ray Scattering and Variational Bayesian Weighting

In-solution synchrotron Small-angle X-ray Scattering (SAXS) technique has been used to investigate an Intrinsically Disordered Protein (IDP) related to Parkinson's disease, the α-synuclein (α-syn), in pre-fibrillar diluted conditions. SAXS experiments have been performed as a function of temperature and concentration on the Wild-Type (WT) and on the three pathogenic mutants G51D, E46K and A53T. To identify the conformers that populate the WT α-syn and the pathogenic mutants in pre-fibrillar conditions, scattering data have been analyzed by a new Variational Bayesian Weighting method (VBWSAS) based on an ensemble of conformers, which includes unfolded monomers, trimers and tetramers, both in helical-rich and strand-rich forms. The developed VBWSAS method uses a thermodynamic scheme to account for temperature and concentration effects and considers long-range protein-protein interactions in the framework of the random phase approximation. The global analysis of the whole set of data indicates that the WT α-syn is mostly present as unfolded monomers and trimers (helical-rich trimers at low T and strand-rich trimers at high T), but not tetramers, as previously derived by several studies. On the contrary, different conformer combinations characterize mutants: in α-syn G51D mutant, the most abundant aggregates at all the temperatures are strand-rich tetramers; strand-rich tetramers are also the predominant forms in the A53T mutant, but their weight decreases with temperature; only monomeric conformers, with a preference for the ones with the smallest sizes, are present in the E46K mutant. The derived conformational behavior then suggests a different availability of species prone to aggregate, depending on mutation, temperature and concentration and accounting for the different neurotoxicity of α-syn variants. Indeed, this approach may be of pivotal importance to describe conformational and aggregational properties of other IPDs.