Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants

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.


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C 5 maleimide and Alexa Fluor 594 C 5 maleimide were used in these reactions. The labelled protein was purified from unreacted dye using the method reported in Cremades et al., 2012 1 .
The reaction yield was checked by mass spectrometry, and proteins labelled less than the 90% were not used in experiments. In order to avoid confusion with unlabelled proteins used in other experiments, labelled A90C, A53T-A90C, A30P-A90C and E46K-A90C will be defined as WT*, A53T*, A30P* and E46K*.
Protein aggregation for Thioflavin T assay. The A53T and A30P pathological aS variants have been reported to have a shorter lag-phase and a longer lag-phase, respectively, than the WT protein for their aggregation reaction in vitro (A53T>WT>A30P) 2 . In order to confirm the different aggregation propensities of the aS mutants are maintained upon the aggregation conditions used for smFRET, a Thioflavin T assay was performed using unlabelled protein, as the fluorescent labels used for the single-molecule fluorescence experiments fluoresce at similar wavelengths to Thioflavin T. WT aS or the Parkinson's disease mutants were prepared from gel filtration purified monomers fractions flash frozen in liquid nitrogen.
Experiments were conducted in triplicate. For each experiments, 600 µL of 70 uM protein in 25 mM Tris-HCl pH 7.4 and 100 mM NaCl were incubated in the dark at 37°C upon 200 rpm orbital shaking (25 mm shaking diameter). Each reaction was supplemented with 0.01 % NaN 3 to prevent bacterial growth. At defined time-points, 10 µl of aggregation mixture were collected and stored at 4°C. Stock solutions of Thioflavin T were prepared in ethanol and the concentration was checked by UV-VIS absorbance using the extinction coefficient of 26,620 M -1 cm -1 at 416 nm. The stock was diluted to 20 µM in the same buffer as the aggregation mixture just before fluorescent measurements. 90 µl of 20 µM Thioflavin T was added to the aggregation aliquot; the mixture was vortexed and then incubated for 30' at room temperature. Measurements were run in a 60 µL volume cuvette with 3 optical quartz windows (Hellma). Fluorescence intensity measurements were recorded with a Cary Eclipse (Varian) accumulating 3 emission spectra from 460 nm to 600 nm. The excitation wavelength was set at 446 nm and the excitation and emission slits were set at 5 nm each. Measurements were taken at 25°C. Curve fitting has been calculated with Origin software considering parameters for Boltzmann equation yielding the least chi squared value. WT aS had an intermediate value for the aggregation lag-phase compared to A53T and A30P ( Figure S1). This indicates that the aggregation propensity is A53T>WT>A30P, in agreement with previously reported works 2 . When compared the kinetics of aggregation for the unlabelled S4 and fluorescently labelled protein variants, we found very similar behaviours and the same relation and trend between the characteristic time for the lag-phase of aggregation for the pathological and WT protein, although the incorporation of the probe is consistently increasing slightly the lag-phase of all the protein variants 1 .
Time-scale of aggregation selection criteria. Before starting the smFRET experiment, it is important to define the time scale of the aggregation reaction, both to check that the aggregation propensities for the mutants is not altered by the labelling and to identify at  Figure S2 shows results for labelled WT (WT*, grey signals), A53T (A53T*, red signals) and A30P (A30P*, blue signals). The decrease in absorbance is slow until a visible pellet is observed (arrows in Figure S2A), at which point it decreased rapidly, indicating that monomers are also able to directly add to fibrils 3 . The experiment was stopped after the deposition of a pellet upon centrifugation but generally two smFRET time-points are analysed after the detection of the protein pellet. There is a small delay compared to the unlabelled protein Thioflavin T assay in Figure S1, but the ability of the protein to aggregate is not affected by the fluorescent tag, as checked by TEM on the pellet formed upon centrifugation ( Figure S3). We then divided the time window for the analysis in two parts: "lag-phase", where no fibrils are present, corresponding to the first days of reaction for WT* and A53T* and the first 48 hours for A30P* (5 time-points for each variant) and "end of the lag-phase", corresponding to the three time-points where the fibrils pellet starts to be visible (30 ± 2 hours, for A53T* at 26 ± 2 hours and for A30P* at 72 ± 16 hours).

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Transmission Electron Microscope imaging. For TEM imaging, WT* labelled with AlexaFluor488 only, or AlexaFluor594 or the equimolar mixture both, were incubated under aggregating conditions. 10 µl of each sample were taken after 48 hours of incubation and applied onto carbon-coated 400-mesh copper grids (Agar Scientific) for 5 min, and then washed with distilled water. Negative staining was carried out by using 2 % (w/v) uranyl acetate. TEM images were acquired using Tecnai G2 microscope (13218, EDAX, AMETEK) operating at an excitation voltage of 200 kV.
Production of microfluidic devices. Microfluidic devices have been prepared as previously surface via spin-coating (800 rpm for 5 s and then ramped to 3000 rpm at an acceleration of 300 rpm/s for 60 s). The final film thickness was 25 µm, measured by profilometry (DekTak 150). After spinning, the wafer was incubated 1 min at 65°C, then 3 min at 95°C and finally 1 min at 65°C, and then exposed to UV light through the mask on a mask aligner (MJB4, SUSS Microtec). After post-baking and development, the master was incubated for 1 min at 170°C. PDMS and curing agent (Sylgard 184, Dow Corning) in a 10/1 w/w ratio were poured over the master, degassed and baked at 75°C at least 2 hours. The devices were separated from the master and cut, using a biopsy punch to introduce access holes for the inlet tubes. The devices were then exposed to oxygen plasma for 7 s (DienerFemto plasma asher), sealed to a glass microscope slide and baked overnight at 75°C.

Calculation of the gamma factor () and association quotient (Q) used for data analysis.
The correction factor (γ) was calculated using a solution of labelled AlexaFluor488 and AlexaFluor594 aS at concentrations enabling the dyes to yield the same absorption at 488 nm (Varian Eclipse Spectrophotometer). The solution was then measured in bulk on the singlemolecule instrument, giving γ = 1.01 according to: where I D and I A are the intensities in the donor and acceptor channels 5 .

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Q is the association quotient used to report the ratio of the rate of coincident events to the sum of the rate of all events in the donor and acceptor channels, subtracting the chance coincident events as indicated 6 : where A and B are the event rates (s -1 ) in donor and acceptor channels and C the observed rate of coincident events, and the estimated rate at which coincident events occur by chance (E) is where is the interval time in seconds. Under these conditions, the number concentration equations can be written as: where Q is the number concentration of oligomers of type-A, k n type-A oligomer formation rate, m tot monomer concentration, k c type-A oligomers conversion rate and P the concentration of type-B oligomers. These can then be solved in closed form, giving the solutions for the number concentrations as follows: where non-seeded initial conditions, Q(0) = P(0) = 0, have been used. Results permit to determine constants k n ' ( = k n m tot nc-1 ) and k c from the number concentrations of type-A and type-B oligomers in the restricted time period consider (data have been fitted till 30 hours for WT*, 26 hours for A53T* and 56 hours for A30P*). The experimental data was fitted to Equations S6 and S7 up to the lag-time and fits were obtained using the two kinetic parameters k n ' and k c . τ lag has been identified through the measure of bulk soluble monomer concentration shown in Figure S2. In order to fit data, data expressed in "fraction of events" have been multiplied by initial monomer concentration (70 µM) to provide an estimation of oligomers molarity ( Figure S4). The results of the fitting are reported in Figure S4 (continuous lines); curves were obtained setting k n ' and k c as 5·10 -8 s -1 and 2·10 -5 s -1 for WT* aS, 4·10 -8 s -1 and 5·10 -5 s -1 for A53T* and 2·10 -8 s -1 and 6·10 -6 s -1 for A30P*.