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

Abstract

We describe here the use of cysteine substitution mutants in the Alzheimer disease amyloid plaque peptide A␤-(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 A␤. The ⌬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 A␤-(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 A␤ 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 ␤-sandwich regions, are accessible to alkylation in the fibril indicating a location in solvent exposed structure.Amyloid fibrils and other non-native protein aggregates are now regarded as an important alternate universe of protein structures formed by normal proteins exposed to conditions of environmental or mutational stress. Fibrils, protofibrils, and other aggregates are associated with a variety of serious human diseases of the brain (1) and periphery (2). Aggregate formation has also long been recognized as a major technical problem in the industrial production and use of proteins (3-6). The intrinsic tendency of polypeptide polymers to generate offpathway aggregates can be viewed as a design flaw in the structural biology of the cell. Since up to 50% of newly synthesized protein chains are aggregated and/or misfolded (7), it is not surprising that major biochemical pathways have evolved in cells and organisms to manage the misfolding and aggregation processes, as exemplified by the chaperone (8), ubiquitin-proteasome (9, 10), aggresome (11), and autophagy (12) systems. In at least a few cases, nature has exploited the ability of polypeptide chains to form amyloid by evolving specific polypeptide sequences and the required cellular machinery for developing functional amyloids, for example as a means of cell attachment (13) or gene regulation (14).Given the growing importance of non-native protein aggregates, it is desirable to gain an increased knowledge of their structures and how th...

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15N relaxation study of the amyloid β-peptide: structural propensities and persistence length

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Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

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