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

Abstract

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-NH(2)) 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 Phe 19 side chains form interstrand contacts in fibrils while Phe 20 side chains interact with the side chain of Va l18. 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 (F(5)-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F(5)-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F(5)-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, F(5)-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 F(5)-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol(-1) for Cha 19,20 and -2.1 kcal mol(-1) for F(5)-Phe 19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.

The Levinthal Problem in Amyloid Aggregation: Sampling of a Flat Reaction Space

“…However, when both mutations are introduced, they actually reduce the growth kinetics, except at very low concentrations (or large τ diff ). These general trends of fibril growth kinetics are fully consistent with experimental results 49 , even though the negative impacts of the double mutation on growth rate appear overestimated. The reason for the overestimation is not entirely clear, though it is likely due to limitations of the protein force field employed.…”

“…Interestingly, the CHA mutations are often non-additive with the double mutation showing a smaller perturbation relative to the WT that one or both of the single mutants. This is a nontrivial prediction that appears consistent with the experimental measurements of fibril growth kinetics 49 . Inspection of Fig.…”

“…Both features are in agreement with existing theoretical and experimental data 47–48 . It has been shown that the Aβ 16-22 sheets further assemble into bilayer or multilayers in solution 24, 49 . As such, the fibril core was represented as a bilayer β-sheet in the current study.…”

Covalent Cross-Linking within Supramolecular Peptide Structures

“…The self-assembled morphologies of peptide nanostructures formed in these experiments are summarized in Figure 2; a detailed account of the various assembly preparations and the results obtained is given as a Supplementary Results section in the Supporting Information. Briefly, transmission electron microscopy (TEM) showed that Aβ 16−22 alone formed pH-dependent nanostructures as has been reported by others, 40,43,45 i.e., filaments at neutral pH and nanotubes at pH 2.0 ( Figure 2, parts a and e). Interestingly, Aβ 16−22 −F19* displayed similar behavior at neutral pH ( Figure 2b; see also the previous work 31 ) but maintained a filamentous morphology at pH 2.0 ( Figure 2f).…”

Melanosomal formation of PMEL core amyloid is driven by aromatic residues

“…Their stability depends on a variety of interactions including hydrogen bonds, electrostatic interactions, hydrophobic contacts, and aromatic π-π stacking34. Amyloids are linked with many incurable diseases including Alzheimer’s, Parkinson’s, and prion diseases.…”