Scanning Cysteine Mutagenesis Analysis of Aβ-(1-40) Amyloid Fibrils

• Published in: The Journal of biological chemistry Vol. 281; no. 2; p. 993
• Main Authors: Shankaramma Shivaprasad, Ronald Wetzel
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 13.01.2006
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M505091200

We describe here the use of cysteine substitution mutants in the Alzheimer disease amyloid plaque peptide Aβ-(1-40) to probe amyloid fibril structure and stabilization. In one approach, amyloid fibrils were grown from Cys mutant peptides under reducing conditions and then challenged with an alkylating agent to probe solvent accessibility of different residues in the fibril. In another approach, monomeric Cys mutants, either in the thiol form or modified with iodoacetic acid or methyl iodide, were grown into amyloid fibrils, and the equilibrium position at the end of the amyloid formation reaction was quantified by determining the concentration of monomeric Aβ. The Δ G values of fibril elongation obtained were then compared in order to provide information on the environment of each residue side chain in the fibril. In general, Cys residues in the N and C termini of Aβ-(1-40) were not only accessible to alkylation in the fibril state but also, when modified in the monomeric state, did not greatly impact fibril stability; these observations were consistent with previous indications that these portions of the peptide are not part of the amyloid core. In contrast, residues 16-19 and 31-34 were not only uniformly inaccessible to alkylation in the fibril state, but their modification with the negatively charged carboxymethyl group in monomeric Aβ also destabilized fibril elongation, confirming other data showing that these segments are likely packed into a hydrophobic amyloid core. Residues 20, 30, and 35, flanking these implicated β-sandwich regions, are accessible to alkylation in the fibril indicating a location in solvent exposed structure.