Clarification of amyloid fibrillation mechanism by modulating intraproteinaceous structure

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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