Extended Data Fig. 3: Interaction between α-synuclein and mammalian proteins.

a, Overlay of two-dimensional [¹⁵N, ¹H]-NMR spectra of 25 µM [U-¹⁵N]-α-synuclein in the absence (grey) and presence (light blue) of 50 µM inhibited HSP90β dimer. Measured in NMR buffer plus 5 mM MgCl₂, 5 mM ATP, 1 µM radicicol and 1 µM geldanamycin. b, Overlay of two-dimensional [¹⁵N, ¹H]-NMR spectra of 100 µM [U-¹⁵N]-α-synuclein in the absence (grey) and presence (light blue) of 200 µM HSC70. c, Overlay of two-dimensional [¹⁵N, ¹H]-NMR spectra of 100 µM [U-¹⁵N]-α-synuclein in the absence (grey) and presence (light blue) of 200 µM HSC70_ADP. Measured in NMR buffer plus 5 mM MgCl₂ and 5 mM ADP. d, Overlay of two-dimensional [¹⁵N, ¹H]-NMR spectra of 100 µM [U-¹⁵N]-α-synuclein in the absence (grey) and presence (light blue) of 200 µM HSC70_ATP. Measured in NMR buffer plus 5 mM MgCl₂ and 5 mM ATP. e, Overlay of two-dimensional [¹⁵N, ¹H]-NMR spectra of 250 µM [U-¹⁵N]-α-synuclein in the absence (grey) and presence (blue) of 500 µM (33 mg ml⁻¹) BSA. f, Overlay of two-dimensional [¹⁵N, ¹H]-NMR spectra of 250 µM [U-¹⁵N]-α-synuclein in the absence (grey) and presence (dark blue) of 500 µM of ubiquitin. g, Residue-resolved combined chemical-shift perturbations of amide moieties upon addition of HSP90β (cyan), inhibited HSP90β (light cyan), HSC70 (light blue), HSC70_ADP (light blue), HSC70_ATP (light blue), BSA (blue) and ubiquitin (dark blue). Broken lines indicate a significance level of two s.d. from the mean. h, Residue-resolved backbone amide NMR signal attenuation (I_rel = I/I₀) of α-synuclein caused by the addition of two equivalents of inhibited HSP90β (light cyan), HSC70 (light blue), HSC70_ATP (light blue) and BSA (blue). i, Residue-resolved NMR signal attenuation (I_rel = I/I₀) of 100 µM [U-¹⁵N]-α-synuclein upon addition of increasing BSA concentrations (50–250 mg ml⁻¹). j, Residue-resolved NMR signal attenuation (I_rel = I/I₀) of 50 µM [U-¹⁵N]-α-synuclein upon addition of increasing ubiquitin concentrations (25–125 mg ml⁻¹). k, Local hydrophobicity of α-synuclein plotted against the amino acid sequence. ΔF are the free energies of transfer of the individual amino acids from an aqueous solution to its surface³⁵. Hydrophobicity corresponds to negative ΔF values. An exponentially weighted seven-window average was applied to the raw data, with the edges contributing 50%. The red line indicates the average value of 1.5 s.d. from the mean, the chosen threshold for the identification of the most hydrophilic segments. l, Sequence-dependent DnaK score for α-synuclein derived from a computational DnaK prediction algorithm³⁶. Regions of the primary sequence with scores less than −5 (red line) are predicted to bind DnaK, a bacterial homologue of HSC70. Experiments in a–f were done in duplicates with similar results.

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