Supporting Information      

Protein-Solvent Interfaces in Human Y145Stop Prion Protein Amyloid Fibrils Probed by

Paramagnetic Solid-State NMR Spectroscopy

Darryl Aucoin,a Yongjie Xia,a Theint Theint,a Philippe S. Nadaud,a Krystyna Surewicz,b Witold K. Surewicz,b and Christopher P. Jaronieca,*

aDepartment of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA

bDepartment of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106, USA

Corresponding author email: jaroniec.1@osu.edu  

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   Figure S1. 2D 15N-13Ca correlation spectra of huPrP23-144 amyloid fibrils (blue contours) and huPrP23-144 fibrils incubated with 20 mM (green contours) and 200 mM Cu(II)-EDTA (red contours). The spectra were recorded at 800 MHz 1H frequency, 11.111 kHz MAS rate and effective sample temperature of ~5 oC, with 15N longitudinal relaxation delays of 10 µs (A-C) and 2 s (D-F) prior to 15N chemical shift encoding.    

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   Figure S2. Residue-specific amide 15N longitudinal relaxation trajectories for huPrP23-144 fibrils (blue circles) and huPrP23-144 fibrils incubated with 20 mM (green circles) and 200 mM Cu(II)-EDTA (red circles). Simulated best-fit trajectories to decaying single exponentials used to determine the longitudinal 15N relaxation rate constants (R1) are shown as solid lines of the corresponding color.

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   Figure S3. 2D 15N-13Ca correlation spectra of huPrP23-144 amyloid fibrils (A; blue contours) and huPrP23-144 fibrils subjected to H/D exchange in D2O-based phosphate buffer for 2 h (B; green contours), 6 h (C; magenta contours) or 48 h (D; red contours). The spectra were recorded at 800 MHz 1H frequency, 11.111 kHz MAS rate and effective sample temperature of ~5 oC as described in the text.        

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     Figure S4. 800 MHz 2D 15N-1H HSQC spectra of huPrP23-144 amyloid fibrils following H/D exchange in D2O-based phosphate buffer and dissolution in 95% (v/v) d6-DMSO, 4.5% (v/v) D2O and 0.5% (v/v) d2-dichloroacetic acid at pD* 5.0, recorded with a total experiment time of 15 min per spectrum. Spectra were recorded with no H/D exchange (blue contours), and 1 h (green contours) and 6 h (red contours) of H/D exchange.      

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   Figure S5. Plots of cross-peak intensity in a series of successive 15 minute 2D 15N-1H HSQC spectra for a representative residue in huPrP23-144 (A133), following H/D exchange in D2O-based phosphate buffer for variable amount of time and dissolution of the amyloid fibrils in 95% (v/v) d6-DMSO, 4.5% (v/v) D2O and 0.5% (v/v) d2-dichloroacetic acid at pD* 5.0. The decrease in the cross-peak intensity over time in the DMSO-based solution is due to the continuing exchange of huPrP23-144 amide protons with the D2O. The cross-peak intensities at time zero were extrapolated from fits of the experimental data to decaying single exponentials, which are shown as solid lines of the corresponding color. The residue-specific peak intensities between spectra recorded for different incubation times in the D2O-based phosphate buffer were also normalized relative to each other as described in the text, to account for any small sample-to-sample variations in protein concentration.        

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   Figure S6. Plots of cross-peak intensities for representative residues in 2D 15N-1H HSQC spectra of huPrP23-144 fibrils following H/D exchange in D2O-based phosphate buffer for variable amounts of time and dissolution in 95% (v/v) d6-DMSO, 4.5% (v/v) D2O and 0.5% (v/v) d2-dichloroacetic acid at pD* 5.0. For each H/D exchange time point the residue-specific cross-peak intensities at time zero prior to fibril dissolution were obtained as described in Figure S5, and the cross-peak intensities (further normalized according to the protein concentration in each sample as described in the text) in the plots are reported relative to the intensities for cross-peaks of the corresponding residues in the reference huPrP23-144 fibril sample that has not undergone any H/D exchange in the D2O-based phosphate buffer.