Clarification of amyloid fibrillation mechanism by modulating intraproteinaceous structure

1. 全二硫缺乏型溶菌酶0SS的淀粉样纤维性颤动反应依赖于盐浓度和pH值，因此蛋白质正电荷的筛选有利于纤维性颤动。它符合成核-延伸反应模式，存在临界单体浓度。核的大小随盐浓度和ph的变化而变化。利用考虑原纤维长度分布的理论方程，得到了单体单体从纤维聚合物末端的解离速率常数的绝对值。在四种1SS变异体中，纤颤率存在着大的，多达三个数量级的变化，它们共同具有各种各样的三级约束和分子内结构，这意味着N端和c端区域应该彼此分开，以便有效地形成原纤维。由SAXS测量得到的原纤维直径…More nt在1SS变异体中差异显著。此外，4SS分子的原纤维在形态和直径上与0SS和1SS分子的原纤维有明显的不同。因此，纤维性颤动的反应速率和机制受到蛋白内结构的种类和数量的强烈调节。通过核磁共振检测的H/ d交换，确定了由于结构形成而不受交换保护的肽区：在0SS纤维中有4个肽区，每个肽区跨越7 - 9个残基，这些残基与高疏水性和β -倾向的区域重合。两个1SS物种的原纤维具有与0SS纤维相似的保护区域，而其他1SS物种的原纤维具有不同的保护区域。此外，这些保护区域与WT溶菌酶原纤维的核心区域完全不同。因此，蛋白内结构的种类和数量调节参与淀粉样蛋白纤颤成核和延伸的肽区。少

1. The amyloid-fibrillation reaction of a lysozyme all-disulfide-deficient variant, 0SS, depends on salt-concentration and pH in such a way that the screening of the protein positive charge facilitates the fibrillation. It conforms to a nucleation-elongation reaction scheme and there exists critical monomer concentrations. A nucleus size varies with varying salt-concentration and pH. The absolute value of the dissociation rate constant of monomeric unit from the end of fibrillar polymer was obtained using a theoretical equation derived for the purpose by taking the length-distribution of fibrils into account. There exists a large, as much as three-orders of magnitude, variation in the fibrillation rate among the four 1SS variants, which collectively have a various kinds of tertiary constraints and intramolecular structures, implying that the N- and C-terminal regions should be apart from each other for efficient protofibril formation. The fibril diameter as obtained from SAXS measureme … More nt differs significantly among the 1SS variants. Moreover, the fibril of a 4SS molecule differs markedly in both morphology and diameter from the fibrils of 0SS and 1SS. Thus, the reaction rate and mechanism of fibrillation are strongly modulated by the kinds and amount of intraprotein structures.2. By using NMR-detected H/D-exchange the peptide regions protected from the exchange by virtue of structure formation were identified : in the 0SS fibril they are four peptide regions, each spanning seven to nine residues, which coincide with the regions of both high hydrophobicity and beta-propensity While the fibrils of two 1SS species show protected regions similar to those of 0SS fibril, the fibrils of the other 1SS species show different protected regions. Moreover, these protected regions altogether differ from the core region of WT lysozyme fibril. Thus, the kinds and amount of intraprotein structure modulate the peptide regions involved in nucleation and elongation of amyloid fibrillation. Less