*2.1. Single Molecule Force Spectroscopy (SMSF)*

The SMFS experiments have been performed on a polyprotein construct containing eight repeats of titin immunoglobulin-like domain (I27) and one grafted AS domain [45–47]. A schematic representation of the polyprotein construct and typical unfolding curves in the absence of ligands are reported in Figure 1A,B.

**Figure 1.** Representative single molecule force spectroscopy (SMFS) recording of α-synuclein (AS) polyprotein and relative statistical analysis. (**A**) Polyprotein construct encompassing the AS full-length polypeptide chain for SMFS experiments. (**B**) Representative force curves of the mechanical unfolding of the polyprotein in distinct conformations stabilized by RC (a), WI (b), and SI (c). Dotted lines are worm-like-chain (WLC) fits to the force-extension curves with free contour length LC and a fixed persistence length Lp = 0.36 nm (see Figure S2 for raw data). Sketches of AS conformations are shown on the right. Diamonds represent weak interactions stabilizing the AS protein, while stars represent strong interactions. (**C**) Statistical distribution of the contour length of the first peak for RC (LC = 79 ± 6 nm), WI (LC = 82 ± 6 nm), and SI (LC = 46 ± 5 nm) conformations. Solid lines represent the Gaussian fits of the histograms. (**D**) Unfolding force statistical distribution of WI (FWI = 117 ± 34 pN) and I27 modules (FI27 = 257 ± 46 pN).

As apparent from Figure 1B, the observed SMFS curves show the typical "sawtooth" pattern, in which the initial part is related to the presence of AS and it is characterized by different mechanical resistances to the unfolding. Each following regular peak is due to the unfolding of an individual I27 domain. Every curve was fitted by means of the worm-like-chain (WLC) model to extract the contour length LC of each peak (both for I27 and AS) [48]. Consistent with the presence of a heterogeneous conformational ensemble, three distinct patterns can be recognized by analyzing the LC of the first peak (Figure 1B,C). A first class of curves displays LC = 79 ± 6 nm (light blue curve, first line of Figure 1B); a second class is characterized by LC = 82 ± 6 nm and by the presence of at least one small peak before the first regular peak (green curve, second line of Figure 1B); a third class displays LC = 46 ± 5 nm and it is characterized by the presence of an additional peak whose height is comparable to the one related to an I27 unfolding event (red curve, third line in Figure 1B). In detail, the light blue curves are ascribed to unstructured conformations of AS and classified as random coil (RC), since no additional peak is detected in the first ~80 nm. The green curves display small (one or more) peaks corresponding to an unfolding force (FWI = 117 ± 34 pN) sensibly lower than I27 (FI27 = 257 ± 46 pN, Figure 1D, Figure S1 and Table S1). These curves are interpreted as representative of a collapsed state of AS mainly stabilized by weak interactions (WI), characterized by an energy barrier to overcome smaller than the one involved in the I27 unfolding. The third and the latter type of curves, characterized by a shorter LC is assigned to a collapsed state of AS, mainly stabilized by strong interactions (SI), which presents resistance to unfolding similar to the one shown by the highly mechanostable protein I27. The extension of the first peak (in the curves assigned to the RC conformation) and that of the first of the higher peaks (in the curves assigned to the WI) are all around 80 nm. These peaks occur when AS is completely extended and flanked by eight I27 folded modules. The measured length is due to the contribution of the eight folded I27 modules (a folded module of I27 is 3 nm long, i.e., 3 nm × 8 = 24 nm), the length of eight linkers between each protein module (a linker is 2 aa, i.e., 8 × 0.36 nm × 2 = 5.76 nm) [47], and the length of the completely extended AS (i.e., 140 aa × 0.36 nm = 50.4 nm). By summing all the contributions, one obtains a total extension of 80.16 nm, which is coherent with the measured values. The subset of curves presenting a LC of the first peak higher than 95 nm, which could be associated with an undesired misfolding event of a I27 module [49], was discarded.
