Probing the Influence of Single-Site Mutations in the Central Cross-β Region of Amyloid β (1-40) Peptides.

Amyloid β (Aβ) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer's disease. A complete mechanistic understanding how Aβ peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aβ 40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe 19 and Leu 34 for cell toxicity. For some mutations at Phe 19 , toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe 19 /Leu 34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aβ assemblies. To this end, we rationally modified positions Phe 20 or Gly 33 . A small library of Aβ 40 peptides with Phe 20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly 33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aβ fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe 20 or Gly 33 led to relatively drastic alterations in the Aβ fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aβ 40 . We suggest that perturbations at position Phe 20 and Gly 33 affect the fibrillation pathway of Aβ 40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe 19 /Leu 34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aβ fibrillation pathway.