Phenylalanine Mutation to Cyclohexylalanine Facilitates Triangular Trimer Formation by ß-Hairpins Derived from Aß.

ABSTRACT

Oligomers of the ß-amyloid peptide, Aß, 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 Aß oligomers. To gain insights into the structure and assembly of Aß oligomers, our laboratory has previously designed and synthesized macrocyclic peptides derived from Aß17-23 and Aß30-36 that fold to form ß-hairpins and assemble to form trimers. In this study, we found that mutating Phe20 to cyclohexylalanine (Cha) in macrocyclic Aß-derived peptides promotes crystallization of an Aß-derived peptide containing the Aß24-29 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 Aß-derived peptides that contain more of the native sequence and thus better mimic the oligomers formed by full-length Aß.