Stabilizing the α-Synuclein dimer

The fibrillation of αSynuclein (αSyn) is a neuropathological marker of Parkinson’s disease (PD). The nucleation of the αSyn fibril is seeded by oligomers. In this context, the structural basis for the first step in oligomerization – dimer formation – is obscure. While dimer formation is an important step, it is one out of many thermodynamically unstable oligomeric species. Therefore, procedures that help stabilize the αSyn dimer distinguished from other αSyn species are needed. Herein, we used multi angle light scattering coupled to anion exchange chromatography (AEX-MALS) for characterizing αSyn Dimers. This combination allows characterization of different species based on molecular weight and ionic strength in parallel. Our results show that αSyn proteins with single Cysteine mutation are stabilized as distinguished monomers and dimers in a MonoQ AEC column. Re-chromatography as well as Ellman`s assay verifies that αSyn monomers and dimers exist in equilibrium, and are not a result of Disulphide Bridge. Repeating the procedure using WT αSyn, the monomers and dimers do not separate, however the single elution peak is associated with an average molecular weight of a mixture of monomers and dimers, proven using size exclusion chromatography with MALS. Therefore, the introduction of cysteines introduces the significant difference in ionic strength between the monomers and the dimers, required for separation in AEC. We will use this procedure to characterize αSyn dimers by comparing crosslinking mass spectrometry (XL-MS) results of αSyn monomers versus dimers, to be later used as spatial restraints in modeling the ensemble structure of αSyn.