Probing aromatic, hydrophobic, and steric effects on the self-assembly of an amyloid-β fragment peptide

Aromatic amino acids have been shown to promote self-assembly of amyloid peptides, although the basis for this amyloid-inducing behavior is not understood. We adopted the amyloid-β 16-22 peptide (Aβ(16-22), Ac-KLVFFAE-NH₂) as a model to study the role of aromatic amino acids in peptide self-assembly. Aβ(16-22) contains two consecutive Phe residues (19 and 20) in which Phe19 side chains form interstrand contacts in fibrils while Phe20 side chains interact with the side chain of Val18. The kinetic and thermodynamic effect of varying the hydrophobicity and aromaticity at positions 19 and 20 by mutation with Ala, Tyr, cyclohexylalanine (Cha), and pentafluorophenylalanine (F₅-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F₅-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F₅-Phe variants self-assembled at dramatically enhanced rates relative to wild-type. Cha mutation was thermodynamically stabilizing at position 20 (ΔΔG = -0.2 kcal mol^-1 relative to wild-type) and destabilizing at position 19 (ΔΔG = +0.2 kcal mol^-1). Conversely, F₅-Phe mutations were strongly stabilizing at both positions (ΔΔG = -1.3 kcal mol^-1 at 19, ΔΔG = -0.9 kcal mol^-1 at 20). The double Cha and F₅-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol^-1 for Cha19,20 and -2.1 kcal mol^-1 for F₅-Phe19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.