Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer's disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe, Phe, and Ile were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe side chains and is clasped by the Ile side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.

A key challenge in studying the biological and biophysical properties of amyloid-forming peptides is that they assemble to form heterogeneous mixtures of soluble oligomers and insoluble fibrils. Photolabile protecting groups have emerged as tools to control the properties of biomolecules with light. Blocking intermolecular hydrogen bonds that stabilize amyloid oligomers provides a general strategy to control the biological and biophysical properties of amyloid-forming peptides. In this paper we describe the design, synthesis, and characterization of macrocyclic β-hairpin peptides that are derived from amyloidogenic peptides and contain the N-2-nitrobenzyl photolabile protecting group. Each peptide contains two heptapeptide segments from Aβ or Aβ constrained into β-hairpins. The N-2-nitrobenzyl group is appended to the amide backbone of Gly to disrupt the oligomerization of the peptides by disrupting intermolecular hydrogen bonds. X-ray crystallography reveals that N-2-nitrobenzyl groups can either block assembly into discrete oligomers or permit formation of trimers, hexamers, and dodecamers. Photolysis of the N-2-nitrobenzyl groups with long-wave UV light unmasks the amide backbone and alters the assembly and the biological properties of the macrocyclic β-hairpin peptides. SDS-PAGE studies show that removing the N-2-nitrobenzyl groups alters the assembly of the peptides. MTT conversion and LDH release assays show that decaging the peptides induces cytotoxicity. Circular dichroism studies and dye leakage assays with liposomes reveal that decaging modulates interactions of the peptides with lipid bilayers. Collectively, these studies demonstrate that incorporating N-2-nitrobenzyl groups into macrocyclic β-hairpin peptides provides a new strategy to probe the structures and the biological properties of amyloid oligomers.

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 3 F20Cha ) and permits elucidation of its structure and assembly by X-ray crystallography. X-ray crystallography shows that peptide 3 F20Cha forms a hexamer. X-ray crystallography and SDS-PAGE further show that trimer 4 F20Cha , a covalently stabilized trimer derived from peptide 3 F20Cha , forms a dodecamer. Size exclusion chromatography shows that trimer 4 F20Cha forms higher-order assemblies in solution. Trimer 4 F20Cha 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.

Antibodies that target the β-amyloid peptide (Aβ) and its associated assemblies are important tools in Alzheimer's disease research and have emerged as promising Alzheimer's disease therapies. This paper reports the creation and characterization of a triangular Aβ trimer mimic composed of Aβ17–36 β-hairpins, and the generation and study of polyclonal antibodies raised against the Aβ trimer mimic. The Aβ trimer mimic is covalently stabilized by three disulfide bonds at the corners of the triangular trimer to create a homogeneous oligomer. Structural, biophysical, and cell-based studies demonstrate that the Aβ trimer mimic shares characteristics with oligomers of full-length Aβ: X-ray crystallography elucidates the high-resolution structure of the trimer and reveals that four copies of the trimer assemble to form a dodecamer; SDS-PAGE, size exclusion chromatography, and dynamic light scattering reveal that the trimer also forms higher-order assemblies in solution; cell-based toxicity assays show that the trimer elicits LDH release, decreases ATP levels, and activates caspase-3/7 mediated apoptosis. Immunostaining studies on brain slices from people who lived with Alzheimer's disease as well as people who lived with Down syndrome reveal that the polyclonal antibodies raised against the Aβ trimer mimic recognize pathological features including different types of Aβ plaques and cerebral amyloid angiopathy. These findings suggest that the triangular trimer structural motif is important in Alzheimer's disease and may thus constitute a new structurally defined molecular target for diagnostic and therapy development.

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