Recombinant mouse prion protein fragment (rMoPrP(89-230)) used in this study was purified and stored as described previously (Milto, Michailova & Smirnovas, 2014 (link)). Protein grade guanidine hydrochloride (GuHCl) was purchased from Carl Roth GmbH, guanidine thiocyanate (GuSCN) and other chemicals were purchased from Fisher Scientific UK.
To prepare different fibril strains, monomeric protein from a stock solution was diluted to a concentration of 0.5 mg/mL in 50 mM phosphate buffer (pH 6) containing 2 M or 4 M GuHCl, and incubated for one week at 37 °C with 220 rpm shaking (in shaker incubator IKA KS 4000i). For seeding experiments rPrP-A4M fibrils were treated for 10 min using Bandelin Sonopuls 3100 ultrasonic homogenizer equipped with MS72 tip (using 20% power, cycles of 30 s/30 s sonication/rest, total energy applied to the sample per cycle—0.36 kJ). The sample was kept on ice during the sonication. Right after the treatment, fibrils were mixed with 0.5 mg/ml of mouse prion solution in 2 M GuHCl in 50 mM phosphate buffer, pH 6, containing 50 µM ThT. Elongation kinetics at 60 °C temperature was monitored by ThT fluorescence assay (excitation at 470 nm, emission at 510 nm) using Qiagen Rotor-Gene Q real-time analyzer (Milto, Michailova & Smirnovas, 2014 (link)). ThT fluorescence curves were normalized by dividing each point by the maximum intensity of the curve.
For denaturation assays, amyloid fibrils were resuspended to a concentration of 25 µM in 50 mM phosphate buffer, pH 6, containing 0.5 M GuSCN and homogenized by sonication (same way as in preparation of seeds). These solutions were diluted 1:4 in a buffer containing varying concentrations of GuSCN, and incubated for 60 min at 25 °C in Maxymum Recovery™ microtubes (Axygen Scientific, Inc., Union City, California, USA). 150 µL of samples were mixed with 850 µL of 100 mM phosphate buffer, pH 7, containing ThT (final concentration after dilution was 50 µM), then each mixture was sonicated for 15 s (same conditions as described above). Fluorescence was measured at 480 nm using the excitation wavelength of 440 nm. Denaturation curves were normalized by dividing each point by the average intensity of the points in the plateau region. Fractional loss of signal at increasing denaturant concentrations corresponds to the fraction of rPrP dissociated from amyloid fibrils.
For AFM experiments, 30 µL of the sample were deposited on freshly cleaved mica and left to adsorb for 1 min, the sample was rinsed with several mL of water and dried gently using airflow. AFM images were recorded in the Tapping-in-Air mode at a drive frequency of approximately 300 kHz, using a Dimension Icon (Bruker, Santa Barbara, California, USA) scanning probe microscope system. Aluminium-coated silicon tips (RTESPA-300) from Bruker were used as a probe.
To prepare samples for the FTIR measurements, rMoPrP aggregates were separated from the buffer by centrifugation (30 min, 15,000 g), and resuspended in D2O, sedimentation and resuspension was repeated three times to minimize the amount of GuHCl and H2O. After resuspension samples were homogenized by 1 min sonication (same conditions as described above). The FTIR spectra were recorded using Bruker Alpha spectrometer equipped with deuterium triglycine sulfate (DTGS) detector. For all measurements, CaF2 transmission windows and 0.1 mm Teflon spacers were used. Spectra were recorded at room temperature. For each spectrum, 256 interferograms of 2 cm−1 resolution were co-added. A corresponding buffer spectrum was subtracted from each sample spectrum. All the spectra were normalized to the same area of amide I/I’ band. All data processing was performed using GRAMS software.
To prepare different fibril strains, monomeric protein from a stock solution was diluted to a concentration of 0.5 mg/mL in 50 mM phosphate buffer (pH 6) containing 2 M or 4 M GuHCl, and incubated for one week at 37 °C with 220 rpm shaking (in shaker incubator IKA KS 4000i). For seeding experiments rPrP-A4M fibrils were treated for 10 min using Bandelin Sonopuls 3100 ultrasonic homogenizer equipped with MS72 tip (using 20% power, cycles of 30 s/30 s sonication/rest, total energy applied to the sample per cycle—0.36 kJ). The sample was kept on ice during the sonication. Right after the treatment, fibrils were mixed with 0.5 mg/ml of mouse prion solution in 2 M GuHCl in 50 mM phosphate buffer, pH 6, containing 50 µM ThT. Elongation kinetics at 60 °C temperature was monitored by ThT fluorescence assay (excitation at 470 nm, emission at 510 nm) using Qiagen Rotor-Gene Q real-time analyzer (Milto, Michailova & Smirnovas, 2014 (link)). ThT fluorescence curves were normalized by dividing each point by the maximum intensity of the curve.
For denaturation assays, amyloid fibrils were resuspended to a concentration of 25 µM in 50 mM phosphate buffer, pH 6, containing 0.5 M GuSCN and homogenized by sonication (same way as in preparation of seeds). These solutions were diluted 1:4 in a buffer containing varying concentrations of GuSCN, and incubated for 60 min at 25 °C in Maxymum Recovery™ microtubes (Axygen Scientific, Inc., Union City, California, USA). 150 µL of samples were mixed with 850 µL of 100 mM phosphate buffer, pH 7, containing ThT (final concentration after dilution was 50 µM), then each mixture was sonicated for 15 s (same conditions as described above). Fluorescence was measured at 480 nm using the excitation wavelength of 440 nm. Denaturation curves were normalized by dividing each point by the average intensity of the points in the plateau region. Fractional loss of signal at increasing denaturant concentrations corresponds to the fraction of rPrP dissociated from amyloid fibrils.
For AFM experiments, 30 µL of the sample were deposited on freshly cleaved mica and left to adsorb for 1 min, the sample was rinsed with several mL of water and dried gently using airflow. AFM images were recorded in the Tapping-in-Air mode at a drive frequency of approximately 300 kHz, using a Dimension Icon (Bruker, Santa Barbara, California, USA) scanning probe microscope system. Aluminium-coated silicon tips (RTESPA-300) from Bruker were used as a probe.
To prepare samples for the FTIR measurements, rMoPrP aggregates were separated from the buffer by centrifugation (30 min, 15,000 g), and resuspended in D2O, sedimentation and resuspension was repeated three times to minimize the amount of GuHCl and H2O. After resuspension samples were homogenized by 1 min sonication (same conditions as described above). The FTIR spectra were recorded using Bruker Alpha spectrometer equipped with deuterium triglycine sulfate (DTGS) detector. For all measurements, CaF2 transmission windows and 0.1 mm Teflon spacers were used. Spectra were recorded at room temperature. For each spectrum, 256 interferograms of 2 cm−1 resolution were co-added. A corresponding buffer spectrum was subtracted from each sample spectrum. All the spectra were normalized to the same area of amide I/I’ band. All data processing was performed using GRAMS software.
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