Depicting Conformational Ensembles of α-Synuclein by Single Molecule Force Spectroscopy and Native Mass Spectroscopy

Description of heterogeneous molecular ensembles, such as intrinsically disordered proteins, represents a challenge in structural biology and an urgent question posed by biochemistry to interpret many physiologically important, regulatory mechanisms. Single-molecule techniques can provide a unique contribution to this field. This work applies single molecule force spectroscopy to probe conformational properties of α-synuclein in solution and its conformational changes induced by ligand binding. The goal is to compare data from such an approach with those obtained by native mass spectrometry. These two orthogonal, biophysical methods are found to deliver a complex picture, in which monomeric α-synuclein in solution spontaneously populates compact and partially compacted states, which are differently stabilized by binding to aggregation inhibitors, such as dopamine and epigallocatechin-3-gallate. Analyses by circular dichroism and Fourier-transform infrared spectroscopy show that these transitions do not involve formation of secondary structure. This comparative analysis provides support to structural interpretation of charge-state distributions obtained by native mass spectrometry and helps, in turn, defining the conformational components detected by single molecule force spectroscopy.

As a consequence of the described unfolding procedure, the several I27 modules of the polyprotein are unfolded under the mechanical action of the applied pulling force. As a result, a series of peaks in the force-extension curve are detected. Each peak is analyzed considering its force maximum value (F U ), which corresponds to the force necessary to unfold a single I27 module. Furthermore, the force-extension data can be fitted to the Worm-Like-Chain (WLC) model in order to extract the asymptotic value (contour length, L C ) for the extension of each I27 module, when the protein chain is completely extended. Figure S1 and Table S1 illustrate the statistical distribution of the F U and L C values for I27 as obtained in the absence or presence of ligands. As shown by the reported data, there is no appreciable change in the F U and L C behavior of I27 due to the addition of DA or EGCG. Thus, the statistical values of F U and L C are collectively evaluated for all the I27 module (F U of I27 in Fig.1D). 28.2 ± 1.1 28.0 ± 0.7 28.1 ± 0.9 28.1 ± 0.9  Table S2 shows the contour length values (mean +/-std) of the first peak of each curve for the three conformations in the presence or absence of ligands. The last column ("Total") reports the cumulative LC values obtained in different conditions and reported in the paper (see Fig.1C).  Fig.1C of the main paper). Table S3 shows the values of the unfolding force (mean +/-std) measured in correspondence of the first peak for the three conformations (RC, SI and WI). The last column ("Total") reports the cumulative F U statistical values, regardless of the different buffer conditions (no ligand, DA, EGCG). The results indicate that ligands do not affect the contour length and the unfolding force of the first peak, although they induce changes in the statistical distributions of conformations.     Figure S5 illustrate the statistical distribution of F U and L C values for the mechanically weak interactions (WI), as obtained under the different tested conditions (no ligand, DA, ECGC). The data indicate that there is no appreciable effect of the ligands on WI F U and L C . As a consequence, the statistical values of F U for WI are collectively evaluated for all the curves classified as WI (Fig.1D of the main paper).   Fig.1D of the main paper).

No
(*) Fig.1D of the main paper).  Each dot of the scatter plot represents a single WI unfolding peak. Data take into account the number of unfolding peaks in each curve: a curve with a single peak contributes with one circle, a curve with two peaks contributes with two squares, a curve with three peaks contributes with three diamonds.  Table S6 :

Number of curves used for analysis of the conformational ensemble Number of curves
Percentage values for SMFS data reported in Fig.2 of the main paper.
To rule out that the conformational differences of (I27) 4 _α-syn_(I27) 4 in the presence of DA or EGCG might be promoted by ligand-induced oxidation rather than ligand binding, the oxidative level of the polyprotein has been analyzed by MS-based proteomics techniques. (I27) 4 _α-syn_(I27) 4 was incubated 4 hours in the absence of ligands and in the presence of either 200 µM DA or 25 µM ECGC, in order to mimic the conditions of SMFS experiments. Then, a fast digestion of the polyprotein was performed by adding trypsin (Sigma-Aldrich, St. Louis, MO) at protease:substrate 1:50 and incubating 30 minutes at 37°C. The reaction was stopped by the addition of 1% formic acid, and the tryptic peptides were desalted by C18 Ziptip (Millipore, Burlington, MA) before injection into an Orbitrap Fusion mass spectrometer coupled to a nano-HPLC system (EASY-nLC 1000, Thermofisher, Waltham, MA). Peptides were separated on a C18 column (length 500 mm, ID 75 µm, particle size 2 µm) by a 90-minutes gradient, analyzed by MS/MS experiments and identified by the software Proteome Discoverer (Thermofisher), using methionine oxidation as a variable modification.
The sequence coverage was at least 92% for each sample run. The I27 domain contains one methionine residue (Met67). Wild-type AS contains four methionine residues (Met1, Met5, Met116 and Met127). However, Met1 has been removed in the construct employed in this work and the C-terminal ones, at positions 116 and 127, are known to be missed in a too large tryptic fragment under these conditions [43]. Thus, I27 Met67 and AS Met5 were used to monitor the oxidation level of the polyprotein. It has been shown that the oxidation kinetics of all AS Met residues is quite similar [43]. The extent of oxidation was quantified by the number of peptide-spectrum matches (PSMs) relative to the total PSMs for the oxidized and non-oxidized variants of each peptide. Both methionine residues feature low oxidation levels after the incubation (below 10% for AS Met5), and no induction by the ligands ( Figure S7). This is in line with the evidence that DA-or EGCG-induced AS oxidation under similar conditions takes place on the time scale of days [43]. Thus, these results confirm that the effects of DA and EGCG revealed in this work by SMFS are ascribable to ligand binding DA or EGCG.

Figure S7
: Percentage of non-oxidized methionine residues in the AS or I27 modules after 4h-incubation of (I27) 4 _α-syn_(I27) 4 polyprotein in the absence of ligands or in the presence of DA or EGCG