File S1 - Diverse Metastable Structures Formed by Small Oligomers of α-Synuclein Probed by Force Spectroscopy

Supporting Experimental Procedures, Supporting References, Figures S1–S8, Table S1. Figure S1. 12% SDS-PAGE gel of α-synuclein samples after purification. Lane 1: monomer; lane 2: dimer; lane 3: tetramer; lane 4: protein ladder. Figure S2. Aggregation into amyloid fibers. α-Synuclein tetramers aggregated over the course of several days to form amyloid fibrils, as seen by the increase in ThT fluorescence. Figure S3. FEC of DNA handles only. A FEC measured on DNA handles only, without protein (black), is well-fit with a WLC model (red). Handle FECs never showed any discrete unfolding transitions like those that occurred when α-synuclein is present. Figure S4. Comparison of CD spectra of monomer, dimer, and tetramer. CD measurements of the α-synuclein monomer (A), dimer (B), and tetramer (C) all show spectra characteristic of unstructured proteins. Figure S5. Examples of unfolding transitions from different structural models. (A) Possible unfolding transitions in a monomer of the β-sandwich model. Unfolding of two β-strands (e.g. β4→β5) produces ΔLc∼8–10 nm. Unfolding all the β-strands in a monomer produces ΔLc∼18 nm. (B) Possible unfolding transitions in a stacked β-sandwich. Unfolding one β-sandwich completely from the tetramer produces ΔLc∼50 nm (upper), while unstacking two β-sandwiches produces ΔLc∼33 nm (lower). (C) Possible unfolding transitions in the α-helical tetramer: unfolding of the N-terminal helix produces ΔLc∼12 nm (top), unfolding the C-terminal monomer produces ΔLc∼33 nm (middle), and unstacking neighboring helix-hairpins produces ΔLc∼18 nm (bottom). Figure S6. Refolding FECs. FECs measured while relaxing the force continuously from the fully-unfolded state did not show any discrete refolding transitions, in contrast to the behavior during unfolding curves, indicating that the structure formation occurs at or near zero force. Figure S7. Effect of waiting time on size-dependent apparent folding rates. The frequency which structures of different size formed in the dimer and tetramer was not noticeably affected when the waiting time at zero force between each pull was changed. In addition to the data shown in Figure 7 are the results for dimers with 0 s waiting time (blue crosses) or over 20 s (blue diamonds) and tetramers with a 10 s waiting time (red stars). Apparent rates were estimated from the occurrence frequency of specific ΔLc values, binned in 15-nm increments to improve the statistics. No clear trend in the number of intermediates formed was observed as a function of the waiting time. Figure S8. Size-dependent folding rate comparison. The apparent rate of formation of structures of different contour lengths in α-synuclein is compared to the folding rate of multi-state natively-folded proteins having different sizes taken from Ivankov et al., 2003. ΔLc values were binned in 5-nm increments for comparison to the rates for natively-folded proteins. Table S1. Summary of potential unfolding distances estimated from structural models in literature.
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