Figure 3

NDGA induces compaction of α-synuclein without preventing structural remodeling. (A) NDGA and mNDGA treatment did not alter α-synuclein collisional cross section measured by IM-MS. (B) cNDGA and cNDGA-5 induced α-synuclein compaction as measured by IM-MS. α-Synuclein was incubated with each molecule at 5:1 molar excess for 10 minutes before measurement. (C) NDGA treatment of α-synuclein did not induce extensive shifts in 2D NMR spectra. α-Synuclein was incubated 1:1 with NDGA for 24 hours before spectra were collected. NDGA-treated α-synuclein spectra (blue) was overlaid on solvent-treated α-synuclein (red). Fluorescein-maleimide (Fam) and tetramethylrhodamide azide (Raz) residue FRET measurement of (D) 0 hour and (E) 24 hour small molecule treatments showed progressive alteration of α-synuclein intramolecular distances by EGCG, but not NDGA, cNDGA, or mNDGA. The dashed line depicts 1 µM α-synuclein treated with buffer (1x PBS). Treatments were 5 µM NDGA (green square), EGCG (red triangle), cNDGA (purple inverted triangle), and mNDGA (blue diamond) (n = 3). (F) α-Synuclein secondary structure was not altered by pretreatment with NDGA. (G) NDGA pretreatment did not prevent α-synuclein assuming α-helical secondary structure in the presence of SDS micelles. α-Synuclein was incubated 1:1 with NDGA, mNDGA, or solvent alone for 24 hours and then dialyzed against PBS for 24 hours. 40 mM SDS micelles were added 5 minutes before analysis. Secondary structure was quantified by circular dichroism. (H) NDGA treatment did not alter α-synuclein phospholipid binding. α-Synuclein was incubated 1:1 with NDGA analogs or solvent alone for 24 hours before fluorescence correlation spectroscopy in the presence of POPS:POPC vesicles at the indicated concentrations (n = 3). (I) Addition of NDGA did not displace fluorescently labeled α-synuclein from POPS:POPC vesicles (n = 3).