Clarification of amyloid fibrillation mechanism by modulating intraproteinaceous structure

1.溶菌酶全二硫缺失突变体0SS的淀粉样原纤化反应依赖于盐浓度和pH，蛋白质正电荷的筛选有利于原纤化。它符合成核-伸长反应模式，存在临界单体浓度。核的大小随盐浓度和pH的不同而不同。利用考虑纤维长度分布的理论公式，得到了单体单元从纤维聚合物末端的离解速率常数的绝对值。四个1SS变体之间的原纤化率存在着高达三个数量级的巨大差异，它们共同具有各种三级限制条件和分子内结构，这意味着为了有效地形成原纤维，N-末端和C-末端区域应该彼此分开。用小角X射线光电子能谱(SAXS)测量了…纤维直径更多的NT在1SS变异体之间存在显著差异。此外，4SS分子的原纤维在形态和直径上都与0SS和1SS的原纤维有明显的不同。因此，蛋白质内结构的种类和数量强烈地影响着原纤化的反应速度和机理。通过核磁共振检测H/D交换，确定了通过结构形成而防止交换的多肽区域：在0SS原纤维中，它们是四个多肽区，每个区域跨度为7-9个残基，与高疏水性和β-倾向性区域相吻合，其中两个1SS种的原纤维显示出与0SS原纤维相似的保护区，而其他1SS种的原纤维显示不同的保护区。此外，这些保护区与WT溶菌酶原纤维的核心区完全不同。因此，蛋白质内部结构的种类和数量调节了与淀粉样蛋白纤颤的成核和延长有关的多肽区域。较少

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