Phenylalanine Mutation To Cyclohexylalanine Facilitates Triangular Trimer Formation By Beta-Hairpins Derived From A Beta

Oligomers of the beta-amyloid peptide, A beta, 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 A beta oligomers. To gain insights into the structure and assembly of A beta oligomers, our laboratory has previously designed and synthesized macrocyclic peptides derived from A beta(17-23) and A beta(30-36) that fold to form beta-hairpins and assemble to form trimers. In this study, we found that mutating Phe(20) to cyclohexylalanine (Cha) in macrocyclic A beta-derived peptides promotes crystallization of an A beta-derived peptide containing the A beta(24-29) loop (peptide 3(F20Cha)) and permits elucidation of its structure and assembly by X-ray crystallography. X-ray crystallography shows that peptide 3(F2ocha )forms a hexamer. X-ray crystallography and SDS-PAGE further show that trimer 4(F20Cha), a covalently stabilized trimer derived from peptide 3(F2ocha), forms a dodecamer. Size exclusion chromatography shows that trimer 4(F2ocha) forms higher-order assemblies in solution. Trimer 4(F2ocha) exhibits cytotoxicity against the neuroblastoma cell line SH-SYSY. These studies demonstrate the use of the F20Cha mutation to further stabilize oligomers of A beta-derived peptides that contain more of the native sequence and thus better mimic the oligomers formed by full-length A beta.