A final buffer composition of 1 M GdnHCl, 3 M urea in 1XPBS

A final buffer composition of 1 M GdnHCl, 3 M urea in 1XPBS, pH 6.0 and the mixture was incubated 37uC with vigorous shaking (around 200?50 r.p.m.). Peptides mPrP(107?43) and mPrP(127?43) were dissolved in deionized water as 100 mM stock solutions. The kinetics of amyloid formation was monitored in SpectraMax Gemini EM (Molecular Devices). Samples containing 50 mM of peptides in presence of 140 mM NaCl and 20 mM NaOAc, pH 3.7 and 10 mM ThT were incubated in 96 well assay plate (Corning, NY) at 25uC without shaking and kinetics was monitored by bottom reading of fluorescence 10457188 intensity at every three hours interval using 445 nm excitation and 487 nm emission. Peptide mPrP(107?26) was dissolved at a concentration of 754 mM in 20 mM HEPES buffer, pH 7.4, 100 mM NaCl, 0.01 NaN3 and dissolution was assisted by sonication. The kinetics was measured at 37uC using SpectraMax Gemini EM as Sudan I described above. All set of experiments were measured in triplicate and subsequent results were expressed as average.Expression and Purification of Full-length mPrP(23?30)For expression and purification of mPrP(23?30), we followed the protocol described by Makarava et al [33]. Briefly, pET101/ D-TOPO-mPrP(23?30), a kind gift from Dr. Ilia V. Baskakov (Center for Biomedical Engineering and Technology, University of Maryland Biotechnology Institute, USA), was transformed into Escherichia coli strain BL21 Star (DE3) (Invitrogen, Carlsbad, California, U.S.A). After induction in the presence of 1 mM IPTG for 5 hr, the cells were harvested, suspended in cell lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 8.0), and subjected to repeated freeze-thaw cycles. Unless stated otherwise, all subsequent steps were conducted at room temperature. TheFigure 1. Amino acid sequences of the prion peptides used. doi:10.1371/journal.pone.0067967.gMouse Prion Amyloid Has Sequence 127?43 in CoreFigure 2. Spontaneous amyloid fibril formation of full length prion Docosahexaenoyl ethanolamide manufacturer protein and prion peptides. Results from three independent measurements (denoted by closed square, ; closed circle, and closed up triangle, m) are shown. (A) mPrP(23?30) (22 mM) in 1 M GdnHCl, 3 M urea in PBS, pH 6.0, was incubated at 37uC with shaking at 220 rpm. (B and D) mPrP(107?43) and mPrP(127?43) (50 mM), respectively in 140 mM NaCl and 20 mM NaOAc, pH 3.7, were incubated at 25uC without shaking. (C) mPrP(107?26) (754 mM) in 100 mM NaCl, 20 mM HEPES, pH 7.4, 0.01 NaN3, was incubated at 37uC without shaking. The kinetics of amyloidogenesis was monitored by ThT binding assay. doi:10.1371/journal.pone.0067967.gNSeed Preparation for the Seeding AssayAmyloid fibrils, generated as described above, were spun down and re-suspended in de-ionized water. The concentration of monomer remaining in solution after the above centrifugation was determined by UV absorption, except for mPrP(107?26), which does not contain Tyr, where the monomer concentration in solution was determined by HPLC. Fibrils generated from peptides or from full-length protein were fragmented using, respectively, 20 or 60 cycles of intermittent pulses (one cycle consists of five pulses of 0.6 sec with a 5 sec interval between two consecutive cycles) with an ultrasonic processor (UP100H, Hielscher, USA) equipped with a 1 mm microtip immersed in the sample. The power during operation was set at 40 . The length of the fragmented fibrils was around 200 nm [34]. To prepare PK-digested mPrP(23?30) seed, the amyloid fibrils were spun down at 15,600 g for 30 mi.A final buffer composition of 1 M GdnHCl, 3 M urea in 1XPBS, pH 6.0 and the mixture was incubated 37uC with vigorous shaking (around 200?50 r.p.m.). Peptides mPrP(107?43) and mPrP(127?43) were dissolved in deionized water as 100 mM stock solutions. The kinetics of amyloid formation was monitored in SpectraMax Gemini EM (Molecular Devices). Samples containing 50 mM of peptides in presence of 140 mM NaCl and 20 mM NaOAc, pH 3.7 and 10 mM ThT were incubated in 96 well assay plate (Corning, NY) at 25uC without shaking and kinetics was monitored by bottom reading of fluorescence 10457188 intensity at every three hours interval using 445 nm excitation and 487 nm emission. Peptide mPrP(107?26) was dissolved at a concentration of 754 mM in 20 mM HEPES buffer, pH 7.4, 100 mM NaCl, 0.01 NaN3 and dissolution was assisted by sonication. The kinetics was measured at 37uC using SpectraMax Gemini EM as described above. All set of experiments were measured in triplicate and subsequent results were expressed as average.Expression and Purification of Full-length mPrP(23?30)For expression and purification of mPrP(23?30), we followed the protocol described by Makarava et al [33]. Briefly, pET101/ D-TOPO-mPrP(23?30), a kind gift from Dr. Ilia V. Baskakov (Center for Biomedical Engineering and Technology, University of Maryland Biotechnology Institute, USA), was transformed into Escherichia coli strain BL21 Star (DE3) (Invitrogen, Carlsbad, California, U.S.A). After induction in the presence of 1 mM IPTG for 5 hr, the cells were harvested, suspended in cell lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 8.0), and subjected to repeated freeze-thaw cycles. Unless stated otherwise, all subsequent steps were conducted at room temperature. TheFigure 1. Amino acid sequences of the prion peptides used. doi:10.1371/journal.pone.0067967.gMouse Prion Amyloid Has Sequence 127?43 in CoreFigure 2. Spontaneous amyloid fibril formation of full length prion protein and prion peptides. Results from three independent measurements (denoted by closed square, ; closed circle, and closed up triangle, m) are shown. (A) mPrP(23?30) (22 mM) in 1 M GdnHCl, 3 M urea in PBS, pH 6.0, was incubated at 37uC with shaking at 220 rpm. (B and D) mPrP(107?43) and mPrP(127?43) (50 mM), respectively in 140 mM NaCl and 20 mM NaOAc, pH 3.7, were incubated at 25uC without shaking. (C) mPrP(107?26) (754 mM) in 100 mM NaCl, 20 mM HEPES, pH 7.4, 0.01 NaN3, was incubated at 37uC without shaking. The kinetics of amyloidogenesis was monitored by ThT binding assay. doi:10.1371/journal.pone.0067967.gNSeed Preparation for the Seeding AssayAmyloid fibrils, generated as described above, were spun down and re-suspended in de-ionized water. The concentration of monomer remaining in solution after the above centrifugation was determined by UV absorption, except for mPrP(107?26), which does not contain Tyr, where the monomer concentration in solution was determined by HPLC. Fibrils generated from peptides or from full-length protein were fragmented using, respectively, 20 or 60 cycles of intermittent pulses (one cycle consists of five pulses of 0.6 sec with a 5 sec interval between two consecutive cycles) with an ultrasonic processor (UP100H, Hielscher, USA) equipped with a 1 mm microtip immersed in the sample. The power during operation was set at 40 . The length of the fragmented fibrils was around 200 nm [34]. To prepare PK-digested mPrP(23?30) seed, the amyloid fibrils were spun down at 15,600 g for 30 mi.