Mapping Aβ Amyloid Fibril Secondary Structure Using Scanning Proline Mutagenesis

Abstract

Although the amyloid fibrils formed from the Alzheimer's disease amyloid peptide Aβ are rich in cross-β sheet, the peptide likely also exhibits turn and unstructured regions when it becomes incorporated into amyloid. We generated a series of single-proline replacement mutants of Aβ(1–40) and determined the thermodynamic stabilities of amyloid fibrils formed from these mutants to characterize the susceptibility of different residue positions of the Aβ sequence to proline substitution. The results suggest that the Aβ peptide, when engaged in the amyloid fibril, folds into a conformation containing three highly structured segments, consisting of contiguous sequence elements 15–21, 24–28, and 31–36, that are sensitive to proline replacement and likely to include the β-sheet portions of the fibrils. Residues relatively insensitive to proline replacement fall into two groups: (a) residues 1–14 and 37–40 are likely to exist in relatively unstructured, flexible elements extruded from the β-sheet-rich amyloid core; (b) residues 22, 23, 29 and 30 are likely to occupy turn positions between these three structured elements. Although destabilized, fibrils formed from Aβ(1–40) proline mutants are very similar in structure to wild-type fibrils, as indicated by hydrogen–deuterium exchange and other analysis. Interestingly, however, some proline mutations destabilize fibrils while at the same time increasing the number of amide protons protected from hydrogen exchange. This suggests that the stability of amyloid fibrils, rather than being driven exclusively by the formation of H-bonded β-sheet, is achieved, as in globular proteins, through a balance of stabilizing and destabilizing forces. The proline scanning data are most compatible with a model for amyloid protofilament structure loosely resembling the parallel β-helix folding motif, such that each Aβ(15–36) core region occupies a single layer of a prismatic, H-bonded stack of peptides.