Structural and Dynamic Insights Into α-Synuclein Dimer Conformations

Abstract

α-Synuclein (αSyn) is an intrinsically disordered protein that forms oligomers and fibrils associated with Parkinson's disease. As such, the mechanism of its oligomerization and its possible links to neurotoxicity have been the focus of many studies. Out of the numerous oligomer types, dimers are the smallest oligomers of aSyn that have been reported. As such, αSyn dimers serve as the earliest steps in the nucleation of αSyn oligomers and later fibrils. Therefore, it is important to characterize αSyn dimers. The identification of αSyn dimers in ensembleaveraged measurements without the use of chemical modifications have been difficult, due to their apparent low abundance. Using analytical anion exchange chromatography coupled to multi angle light scattering as well as to dynamic light scattering, we show that recombinant αSyn is in equilibrium between different types of monomers and compact dimers, and that both are abundant. Additionally, bulk Förster resonance energy transfer (FRET), fluorescence cross-correlation spectroscopy (FCCS) of FRET and pulsedinterleaved excitation single-molecule FRET (PIE smFRET) measurements of mixtures of donor-and acceptor-labeled αSyn. These measurements indicated a dimer dissociation constant of 1.75 μM. We concluded that αSyn dimers exist as abundant species in equilibrium with monomers only if produced to reach concentrations of hundreds of nanomolar or above.

Big versus small: The impact of aggregate size in disease

“…DMD simulations are combined with various experimental technologies, such as FRET, Mass Spectrometry, which have been demonstrated by previous studies (J. Chen, Zaer, et al, 2021;Proctor et al, 2016;Zamel et al, 2019). Protein structures predicted by DMD simulations that are guided and validated by experimental data are more accurate than those predicted solely by computational methods (J.…”

“…By integrating cross-linking mass spectrometry with DMD simulations, the same group utilized cross-linking data as constraints to simulate the dimer structures of α-synuclein. They identified one compact and stable dimer structure where tyrosine 39 is sufficiently close to forming a dityrosine covalent bond, which may be involved in α-synuclein amyloid fibril formation (Zamel et al, 2019).…”

Generating Ensembles of Dynamic Misfolding Proteins

“…Using lasers or LEDs the timescale needed for photo-crosslinking can be reduced from tens of minutes to less than seconds ( Russmann et al, 1998 ), providing a clearer snap-shot of the interactions by reducing averaging ( Horne et al, 2018 ). These experiments capture the dynamic nature of intra/inter-molecular contacts and combined with computational analysis can visualize these species ( O’Reilly and Rappsilber, 2018 ; Zamel et al, 2021 ). Even though in some cases crosslinking restraints have been treated as NMR-derived distances, care has to be taken when dealing with ensembles of structures, since the nature of the two distances is fundamentally different.…”