Supplementary Figure 1

Supplementary Figure 1: Characterization of the interactions between STI1 and α-synuclein by NMR titration. a. 600 MHz 1H-15N HSQC spectrum of 100 µM α-synuclein (in 20 mM Hepes, 50 mM NaCl, pH 7.2) in the absence (black) and the presence (green) of 200 µM of STI1 TPR1 domain. b. 600 MHz 1H-15N HSQC spectrum of 100 µM α-synuclein (in 20 mM Hepes, 50 mM NaCl, pH 7.2) in the absence (black) and the presence (cyan) of 200 µM of STI1 TPR2B domain. c. NMR titration of unlabeled STI1 to 15N-labeled α-synuclein. The concentration of α-synuclein was kept constant at 100 µM (estimated by Lowry assay) while the concentrations of STI1 varied from 0 µM to 200 µM in increments of 25 µM (estimated by Lowry assay). The KD value was determined based on the titration data of D119, D121, and E137, the three residues that displayed the largest chemical shift changes upon the addition of STI1. Protein concentrations determined by amino acid analysis were used in fitting the KD value. d. NMR titration of unlabeled TPR2A to 15N-labelled α-synuclein. The concentration of α-synuclein was kept constant at 100 µM (estimated by Lowry assay) while the concentrations of TPR2A varied from 0 µM to 200 µM (estimated by Lowry assay) in increments of 25 µM. The KD value was determined based on the titration data of D119, D121, and E137, the three residues that displayed the largest chemical shift changes upon the addition of TPR2A. Protein concentrations determined by amino acid analysis were used in fitting the KD value. e. Overlay of 1H-15N HSQC spectra of 100 µM 15N-labelled α-synuclein (black), 100 µM 15N-labelled α-synuclein + 100 µM TPR2A (red), and 100 µM 15N-labeled α-synuclein + 100 µM TPR2A + 100 µM Hsp90 pentapeptide (green).