Phenylalanine Mutation to Cyclohexylalanine Facilitates Triangular Trimer Formation by β‑Hairpins Derived from Aβ

Oligomers
of the β-amyloid peptide, Aβ, play a central
role in the pathogenesis and progression of Alzheimer’s disease.
Trimers and higher-order oligomers composed of trimers are thought
to be the most neurotoxic Aβ oligomers. To gain insights into
the structure and assembly of Aβ oligomers, our laboratory has
previously designed and synthesized macrocyclic peptides derived from
Aβ17–23 and Aβ30–36 that fold to form β-hairpins and assemble to form trimers.
In this study, we found that mutating Phe20 to cyclohexylalanine
(Cha) in macrocyclic Aβ-derived peptides promotes crystallization
of an Aβ-derived peptide containing the Aβ24–29 loop (peptide 3F20Cha) and
permits elucidation of its structure and assembly by X-ray crystallography.
X-ray crystallography shows that peptide 3F20Cha forms a hexamer. X-ray crystallography and SDS-PAGE
further show that trimer 4F20Cha, a covalently stabilized trimer derived from peptide 3F20Cha, forms a dodecamer. Size exclusion
chromatography shows that trimer 4F20Cha forms higher-order assemblies in solution. Trimer 4F20Cha exhibits cytotoxicity against the
neuroblastoma cell line SH-SY5Y. These studies demonstrate the use
of the F20Cha mutation to further stabilize oligomers of Aβ-derived
peptides that contain more of the native sequence and thus better
mimic the oligomers formed by full-length Aβ.