Abstract
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.