Conformation and fibrillogenesis of Alzheimer Aβ peptides with selected substitution of charged residues

A key pathological feature of Alzheimer's disease (AD) is the formation and accumulation of amyloid fibers within the neurophil as senile placques and in the walls of cereb ral and meningeal blood vessels. The major component is the 39 to 42 residue amyloid P protein (AP), which is an internal proteolytic fragment of the membrane-associated amyloid precursor protein. Aggregation of AP into amyloid fibers that could be cytotoxic may be a factor in the AD-related neuronal loss. To understand the steps and molecular interactions involved in the transition from a soluble to fibrous form of Aβ, and to test molecular models that postulate ion pairing between β-strands, we have synthetized four peptides having substitutions in specific, charged residues. These included an Aβ fragment, residues 11 to 25, and having histidine-to-aspartate replacements at positions 13 (H13D) and 14 (H14D), an aspartate-to-lysine at position 23 (D23K) and a 28-mer full-length extracellular domain where the positive charge cluster at His13-His14-Qln15-Lys16 was replaced by an uncharged Gly13-Gly14-Gln15-Gly16 (GGQG). Fourier-transform infrared spectroscopy and fiber X-ray diffraction determined that the H13D and H14D substitutions had negligible effect on β-sheet formation, suggesting that these residues are not critical for the intramolecular interactions necessary for folding in the β-conformation. However, negative-stain electron microscopy revealed that the loss of the His13 or His14 resulted in only protofilament formation, suggesting that these residues are involved in amyloid fibril assembly. By contrast, the D23K substitution virtually eliminated folding into a β-sheet conformation, with appreciable secondary structure being detected only following extended incubation times. The complete absence of the centrally charged region GGQG arrested amyloid assembly at the protofilament stage and also reduced the stability of the β-conformation, suggesting a contribution; of Lys 16 in maintaining secondary structure. While it has been conclusively demonstrated by previous investigations that amyloid formation is dependent to a large extent on hydrophobically driven interactions, our results indicate that charge-charge interactions function in concert with non-ionic interactions to stabilize the β-sheet conformation and assembly of AD amyloid fibers.