Abstract
Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson’s disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.
Highlights
A53T variant suggest that the protein presents an increased propensity to acquire beta-sheet structure when the Alanine residue at position 53 is mutated to Threonine[19,24,25,26,27]; effect that has been correlated with the faster apparent aggregation rate of this mutant obtained experimentally[16,28,29]
We report here the detection and characterization of A53T*, A30P* and E46K* aS oligomers, and the comparison of them with oligomers formed from WT* aS, both in terms of their kinetics of formation, interconversion and accumulation, as well as in terms of their structural properties
Together with previous studies reporting the loss of a propensity to form secondary structure for this mutant[19,22,24] and to the well-established observation that Pro residues break the tendency of the protein to acquire beta-sheet structure[20,21], our study suggests that residue 30 and/or its neighbouring residues are important for the misfolding and assembly of the protein into both type-A and type-B oligomeric species
Summary
A53T variant suggest that the protein presents an increased propensity to acquire beta-sheet structure when the Alanine residue at position 53 is mutated to Threonine[19,24,25,26,27]; effect that has been correlated with the faster apparent aggregation rate of this mutant obtained experimentally[16,28,29]. The characterization of the oligomeric species generated during the lag-phase before fibril formation has been demonstrated to be challenging due to their intrinsic transient, heterogeneous and low abundant nature Their identification and characterization during the aggregation reaction has been primarily derived from either indirect experimental data[16,28,34] or by means of enriching the samples in certain types of highly stable oligomeric species[29,36,37,38]. We have incorporated fast-flow microfluidic techniques to the single-molecule fluorescence technique in order to increase the rate of data acquisition and ameliorate effects from the free diffusion of particles through the edges of the confocal volume[48]; this approach has been recently validated and their application has allowed us to obtained more detailed information of the nature of the different amyloid oligomeric species formed[49]. The type-B oligomers formed from the A53T and A30P mutants had different FRET efficiencies to those formed from WT aS, indicating structural variations in the type-B oligomeric ensemble depending on the protein variant
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