Alanine Scanning Mutagenesis of Aβ(1-40) Amyloid Fibril Stability

We describe here an alanine scanning mutational analysis of the Aβ(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15–36, leads to destabilization of the elongation step, compared to wild-type, by about 1 kcal/mol, consistent with a major role for hydrophobic packing in Aβ(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel β-sheet in the small, globular protein Gβ1. We utilize these Ala-WT ΔΔG values to filter previously described Pro-WT ΔΔG values, creating Pro-Ala ΔΔG values that specifically assess the sensitivity of a sequence position, in the structural context of the Aβ fibril, to replacement by proline. The results provide a conservative view of the energetics of Aβ(1-40) fibril structure, indicating that positions 18–21, 25–26, and 32–33 within amyloid structure are particularly sensitive to the main-chain disrupting effects of Pro replacements. In contrast, residues 14–17, 22, 24, 27–31, and 34–39 are relatively insensitive to Pro replacements; most N-terminal residues were not tested. The results are discussed in terms of amyloid fibril structure and folding energetics, in particular focusing on how the data compare to those from other structural studies of Aβ(1-40) amyloid fibrils grown in phosphate-buffered saline at 37 °C under unstirred (“quiescent”) conditions.