Exploring and controlling the supramolecular assembly of amyloid-forming peptides and proteins with chemical model systems

Chapter 1 overviews the phenomenon of the self-assembly of small peptides and proteins in several neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. This chapter provides context for the rest of the dissertation. The aberrant assembly of peptides and proteins into large structures defines a class of human pathologies, which are collectively known as amyloid diseases. In these diseases, native peptides and proteins misfold and proceed to assemble into structures that mediate the disease. The characterization of these assemblies is particularly challenging due to their heterogeneity and metastability. Within this cornucopia of assemblies, a subset known as soluble oligomers have emerged as likely neurotoxic species responsible for the progression of neurodegenerative diseases. The structures of these oligomers remain unknown. This dissertation describes my efforts to explore and control the structures of these oligomers with chemical model systems.Chapter 2 presents the X-ray crystallographic structure and biological characterization of oligomers formed by a macrocyclic β-hairpin peptide derived from α-synuclein. The peptide adopts a β-hairpin structure, which assembles in a hierarchical fashion. Three β-hairpins assemble to form a triangular trimer. Three copies of the triangular trimer assemble to form a basket-shaped nonamer. Two nonamers pack to form an octadecamer. Molecular modeling suggests that full-length α-synuclein may also be able to assemble in this fashion. Circular dichroism spectroscopy demonstrates that the peptide interacts with anionic lipid bilayer membranes, like oligomers of full-length α-synuclein. LDH and MTT assays demonstrate that the peptide is toxic toward SH-SY5Y cells. Comparison of the peptide to homologues suggests that this toxicity results from nonspecific interactions with the cell membrane. The oligomers reported are fundamentally different than the proposed models of the fibrils formed by α-synuclein and suggest that α-Syn36–55, rather than the NAC, may nucleate oligomer formation.Chapter 3 explores the effect of shifting the residue pairing of Aβ-derived β-hairpins on the structures of the oligomers that form through X-ray crystallography. Three residue pairings were investigated using constrained macrocyclic β-hairpins in which Aβ30–36 is juxtaposed with Aβ17–23, Aβ16–22, and Aβ15–21. X-ray crystallography reveals that the Aβ16–22–Aβ30–36 pairing forms a compact ball-shaped dodecamer composed of fused triangular trimers, the Aβ17–23–Aβ30–36 forms a spherical dodecamer composed of triangular trimers, and that the Aβ15–21–Aβ30–36 pairing forms a fibril-like assembly. Both the compact dodecamer and the spherical dodecamer may help explain the structures of the trimers and dodecamers formed by full-length Aβ.Chapter 4 describes the design, synthesis, and characterization of macrocyclic β-hairpins that contain the N-2-nitrobenzyl photolabile protecting group. Each peptide contains two heptapeptide segments from Aβ16–22 or Aβ17–23 constrained into β-hairpins. The N -2-nitrobenzyl group is appended to the amide backbone of Gly33 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.Chapter 5 presents the discovery that crystal violet and other C3 symmetric triphenylmethane dyes bind to triangular trimers derived from Aβ. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This chapter 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 Phe19, Phe20, and Ile31 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 three Phe20 side chains and is clasped by the side chains of Ile31. 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