Supporting Information

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SI Methods

The ORF of the mouse PrP gene was amplified by PCR using oligonucleotide primers designed to create a specific 5' CACC end for directional cloning of the fragment into the expression vector pET101/D-TOPO (Invitrogen). The pET101/mPrP23-231 construct was used to transform the Escherichia coli host strain BL21 Star (DE3) (Invitrogen). A single colony on a Luria-Bertani (LB) agar plate with 0.1 mg/ml carbenicilin was picked and inoculated into 2 ml of LB medium containing 0.1 mg/ml carbenicilin.

After overnight growth at 37°C with shaking, the transformants were inoculated into 40 ml of Spectra9 minimum medium (>98% ¹⁵N; Spectra Gases) supplemented with 0.1 mg/ml ampicillin, and incubation continued for a further 8 h. The entire culture was inoculated into 1 liter of Spectra9 medium containing 0.1 mg/ml carbenicilin and grown at 37°C with shaking for 4 h. After OD₆₀₀ reached 1.0, expression was induced by addition of isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and the culture was incubated for an additional 10 h.

Cells were harvested by centrifugation, and 4.3 g of the pellet was resuspended in 37 ml of lysis buffer (50 mM Tris, pH 8/1 mM EDTA/100 mM NaCl), followed by the addition of 0.2 mg/ml lysozyme and 20 mg/ml phenylmethanesulfonyl fluoride (PMSF), with subsequent incubation of 1 h on ice. Then, 1 mg/ml deoxycholic acid and 10 mg/ml DNase I were added, and the mixture incubated for 2.5 h. Centrifugation at 20,000 ´ g for 20 min pelleted the insoluble materials. Solubilization of the protein in the pellet was achieved by resuspension in 23.5 ml of loading buffer (100 mM sodium phosphate/10 mM Tris, pH 8.0/8 M urea/10 mM 2-mercaptoethanol) and incubation at room temperature for 2 h (1). The remaining insoluble materials were pelleted by a further centrifugation at 20,000 ´ g for 80 min.

The entire supernatant was loaded onto a 1-ml Ni-chelating affinity column (Amersham Pharmacia Biosciences) freshly Ni-loaded and equilibrated with loading buffer. After washing the column with 7 ml of the same buffer, ¹⁵N-labeled recombinant PrP was recovered with elution buffer [100 mM sodium phosphate/10 mM Tris, pH 4.5/8 M urea). Fractions containing the recombinant protein were pooled and diluted with deionized water and the buffer [10 mM Tris, pH 8.0/4 M urea) to a final concentration of 0.1 mg/ml protein and 4 M urea. The pH of the sample solution was adjusted to 8.0 by the addition of a tiny amount of concentrated NaOH solution.

After incubation at room temperature for 2 days, completion of the oxidization of the protein was confirmed by the analytical reversed-phase HPLC (2) with a 4.6 mm ´ 25 cm C4 column (COSMOSIL 5 C4-AR-300; Nacalai Tesque). The ¹⁵N-labeled PrP was concentrated, and the buffer was replaced for NMR measurement by ultracentrifugation using an Amicon Ultrafree-CL (NMWL 5,000) filter (Millipore) to a final protein concentration of 1.2 mg/ml. The concentration of mouse PrP was estimated by the absorbance at 280 nm using the specific absorbance (3) of e₂₈₀ = 2.7 (mg/ml)^-1·cm^-1.

Interaction between prion protein and compounds was analyzed using the BIAcore T100 system. A recombinant full-length mouse prion protein (recombinant PrP) was immobilized on a sensor chip (CM5) according to the manufacturer's instructions. Various concentrations of WS-GN8 were injected into running buffer (0.005% Surfactant P20 in HBS, pH 7.4) for 1 min at a flow rate of 30 ml/min; then running buffer without GN8 was injected for 1 min at the same flow rate. Data were corrected by using a blank sensor chip as a control.

From the plot the response of SPR, RU against the concentration of GN8 (circle in SI Fig. 5), we initially estimated the contribution of the nonspecific binding at higher concentration of GN8, which goes up linearly with the concentration of GN8 (solid line). Specific binding at low concentration of GN8 was extracted by subtraction of the nonspecific contribution (dotted line) from the total response. Specific binding component showed the saturation, and the Scatchard plot (Req vs. Req/C, where Req and C are the equilibrium response of SPR and the concentration of GN8, respectively) shown in Fig. 2a indicated the strong specific interaction between PrP^C and GN8. From the slope of the Scatchard plot, its dissociation constant (K_d) was calculated to be 3.9 mM.

All of the circular dichroism measurements were acquired by using an Aviv 202 stopped-flow circular dichroism spectrometer. Far-UV spectra were recorded between 180 and 250 nm by using a cell (1-nm slit width and 1-mm path length) thermostated at 25°C. Thermal unfolding of mouse recombinant PrP was monitored by measuring the CD signal at 222 nm of samples containing 5 mM PrP with or without 10 mM candidate compounds at a temperature range of 25-85°C, with a heating rate of 1°C/min. All of the measurements were done in 100 mM sodium phosphate buffer at pH 7.4. To verify that the T_m values were not dependent on the heating rate, samples were thermally unfolded at heating rates of 2, 1, and 0.5°C/min. The thermal unfolding profiles were fitted to a two-state model (4):

,

where R and T are the gas constant and the temperature (in K), respectively; T_m is the melting temperature; and DH is the enthalpy change associated with thermal unfolding. Y is the CD signal at 222 nm, and Y_N and Y_U are the signals contributed by the native and unfolded states, respectively. m_N and m_U indicate the slope of native and unfolded states, respectively. We used a nonlinear least-square fit routine of the program SigmaPlot 2001 (SPSS, Chicago, IL).

1. Bocharova OV, Breydo L, Parfenov AS, Salnikov VV, Baskakov IV (2005) J Mol Biol 346:645-659.

2. Lu BY, Beck PJ, Chang JY (2001) Eur J Biochem 268:3767-3773.

3. Hornemann S, Korth C, Oesch B, Riek R, Wider G, Wuthrich K, Glockshuber R (1997) FEBS Lett 413:277-281.

4. Ramsay GD, Eftink MR (1994) Methods Enzymol 240:615-645.