Rements), in the presence of 30 mL of mPrP(23?30) seed (denoted by closed, half-filled and open circles for three independent measurements), or in the presence of the same amount of mPrP(23?30) seed digested by proteinase K (denoted by closed, half-filled and open up triangles for three independent measurements). doi:10.1371/journal.pone.0067967.ganalyze morphology. As shown in Figure S3, all three peptidegenerated fibrils remained intact under denaturing condition, ruling out the possibility of poor stability. In contrast, amyloidogenesis of mPrP(23?30) was induced immediately on addition of only 20 mL of sonicated mPrP(127?43) seed containing only 44 pmoles of mPrP(127?43) per microliter seed solution (Figure 6B), a seeding effect similar to that seen with mPrP(107?43) seed in Figure 4B. Our results showed that, though peptide mPrP(107?143) can seed full-length recombinant prion protein, the seeding ability resides in 16574785 the Title Loaded From File C-terminal segment of this peptide.DiscussionThe in vitro formation of amyloid fibril from T the effect of PEITC was more pronounced in HER2 positive soluble monomeric recombinant prion protein provides an insight into the structural conversion of prion protein, which ultimately leads to amyloidogenesis. With regard to the structure of soluble prion protein, it is important to locate the regions, which take part in the conversion process. According to several models, the process of b-aggregation starts when segments that possess high hydrophobicity, a high bsheet propensity, and low net charge become exposed to the solvent and can associate [40?3]. Hydrophobicity analysis of the prion protein sequence revealed the existence of three hydrophobic clusters, one in the region of amino acids 110?37 and the other two reside in helices 2 and 3 [21]. The N-terminal half of mPrP(107?43), i.e. mPrP(107?26), formed spontaneous amyloid fibrils, though with a considerable lag phase. This is in agreement with the findings reported by Gasset et al [15]. One interesting point is that this peptide needed a much higher monomer concentration (754 mM) for initiation of fibril formation, but the monomer concentration remained in solution after fibrillization was only 12.4, 11.1 and 6.6 mM in three independent experiments. In contrast, the C-terminal half of mPrP(107?43), i.e. mPrP(127?143), underwent fibrillization without any detectable lag time for nucleus formation at a peptide concentration of 50 mM but the monomer concentration remained in solution after fibrillization was 32.6, 35.6 and 27.2 mM in three independent experiments. Our data suggested that (1) mPrP(127?43) might contain an intrinsic structural element that drives nucleation; (2) mPrP(127?143) has a higher thermodynamic solubility than mPrP(107?26);and (3) mPrP(107?26) might have a much higher energy barrier in the nucleation step. In this connection it is worth to mention that only having high hydrophobicity does not ensure a peptide segment of a protein to act as nucleation site where amyloidogenesis can begin. This notion is supported by the fact that in spite of having high hydrophobicity mPrP(107?26) requires high monomer concentration probably to overcome a high energy barrier during nucleation. In 23977191 order to locate potential sites of nucleation which can act as amyloidogenic hot-spots we utilized two bioinformatic prediction methods, namely, FoldAmyloid [44] and Aggrescan [45], which use amino acid composition of proteins as the basic approach for assigning amyloidogenic hot-spots. Prediction from both the methods revealed.Rements), in the presence of 30 mL of mPrP(23?30) seed (denoted by closed, half-filled and open circles for three independent measurements), or in the presence of the same amount of mPrP(23?30) seed digested by proteinase K (denoted by closed, half-filled and open up triangles for three independent measurements). doi:10.1371/journal.pone.0067967.ganalyze morphology. As shown in Figure S3, all three peptidegenerated fibrils remained intact under denaturing condition, ruling out the possibility of poor stability. In contrast, amyloidogenesis of mPrP(23?30) was induced immediately on addition of only 20 mL of sonicated mPrP(127?43) seed containing only 44 pmoles of mPrP(127?43) per microliter seed solution (Figure 6B), a seeding effect similar to that seen with mPrP(107?43) seed in Figure 4B. Our results showed that, though peptide mPrP(107?143) can seed full-length recombinant prion protein, the seeding ability resides in 16574785 the C-terminal segment of this peptide.DiscussionThe in vitro formation of amyloid fibril from soluble monomeric recombinant prion protein provides an insight into the structural conversion of prion protein, which ultimately leads to amyloidogenesis. With regard to the structure of soluble prion protein, it is important to locate the regions, which take part in the conversion process. According to several models, the process of b-aggregation starts when segments that possess high hydrophobicity, a high bsheet propensity, and low net charge become exposed to the solvent and can associate [40?3]. Hydrophobicity analysis of the prion protein sequence revealed the existence of three hydrophobic clusters, one in the region of amino acids 110?37 and the other two reside in helices 2 and 3 [21]. The N-terminal half of mPrP(107?43), i.e. mPrP(107?26), formed spontaneous amyloid fibrils, though with a considerable lag phase. This is in agreement with the findings reported by Gasset et al [15]. One interesting point is that this peptide needed a much higher monomer concentration (754 mM) for initiation of fibril formation, but the monomer concentration remained in solution after fibrillization was only 12.4, 11.1 and 6.6 mM in three independent experiments. In contrast, the C-terminal half of mPrP(107?43), i.e. mPrP(127?143), underwent fibrillization without any detectable lag time for nucleus formation at a peptide concentration of 50 mM but the monomer concentration remained in solution after fibrillization was 32.6, 35.6 and 27.2 mM in three independent experiments. Our data suggested that (1) mPrP(127?43) might contain an intrinsic structural element that drives nucleation; (2) mPrP(127?143) has a higher thermodynamic solubility than mPrP(107?26);and (3) mPrP(107?26) might have a much higher energy barrier in the nucleation step. In this connection it is worth to mention that only having high hydrophobicity does not ensure a peptide segment of a protein to act as nucleation site where amyloidogenesis can begin. This notion is supported by the fact that in spite of having high hydrophobicity mPrP(107?26) requires high monomer concentration probably to overcome a high energy barrier during nucleation. In 23977191 order to locate potential sites of nucleation which can act as amyloidogenic hot-spots we utilized two bioinformatic prediction methods, namely, FoldAmyloid [44] and Aggrescan [45], which use amino acid composition of proteins as the basic approach for assigning amyloidogenic hot-spots. Prediction from both the methods revealed.
