UC Irvine

Chapter 1 provides a brief review on the application and use of peptide model systems to mimic the properties and structures of oligomers formed by amyloidogenic peptides and proteins. Oligomers formed by amyloidogenic peptides and proteins are noted for the significant heterogeneity they display in their stability, structure, and stoichiometry. Obtaining high-resolution structures of the oligomers formed by amyloidogenic peptides and proteins is crucial for advancing our knowledge of the molecular basis of the diseases these assemblies are associated with. The assembly of amyloidogenic peptides and proteins such as the β-amyloid peptide (Aβ), α-synuclein, huntingtin, tau, and islet amyloid polypeptide (IAPP) into amyloid fibrils and oligomers is directly linked to amyloid diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, frontotemporal dementias, and type II diabetes. Although amyloid oligomers have emerged as especially important in amyloid diseases, high-resolution structures of the oligomers formed by full-length amyloidogenic peptides and proteins have remained elusive. Investigations of oligomers assembled from fragments or stabilized β-hairpin segments of amyloidogenic peptides and proteins have allowed investigators to illuminate some of the structural, biophysical, and biological properties of amyloid oligomers This chapter provides context for much of the work described in this dissertation and highlights recent advances in the study of amyloidogenic oligomers and challenges currently faced by the field. To mimic and investigate the structures, biological, and biophysical properties of endogenous Aβ oligomers, the Nowick laboratory has developed macrocyclic β-hairpin peptides as model systems. These macrocyclic β-hairpin peptides comprise β-strand segments of residues that are derived from the full-length sequence of Aβ. To constrain the peptide to a macrocycle and stabilize a β-hairpin conformation, we use disulfide bridges and δOrn turn units. To prevent the uncontrolled aggregation of these Aβ derived peptide our group typically incorporates an N-methyl group on one β-strand. In studying the assembly of these Aβ derived macrocyclic β-hairpin peptides, our laboratory has been able to report several crystallographic structures of dimers and trimers that further assemble to form tetramers, hexamers, octamers, and dodecamers. We believe that these unique structures reflect some of the immense variation and heterogeneity present in the structures of endogenous Aβ oligomers. This thesis describes my efforts to build upon the archetypal macrocyclic β-hairpin model system developed by the Nowick laboratory to better mimic endogenous Aβ oligomers. In the course of my doctoral studies, I have developed and studied three new macrocyclic β-hairpin model systems. These model systems incorporate additional residues beyond the Aβ16–36 residue segments typically studied by our laboratory and explore the incorporation of α-methyl groups in lieu of N-methyl groups to limit uncontrolled aggregation. These new macrocyclic β-hairpin model systems have also provided high-resolution insight into the unique ways in which Aβ β-hairpins can assemble to form oligomers. Chapter 2 describes the synthesis, solution-phase biophysical studies, and X-ray crystallographic structures of hexamers formed by macrocyclic β-hairpin peptides derived from the central and C-terminal regions of Aβ, which bear “tails” derived from the N-terminus of Aβ. Soluble oligomers of Aβ, are thought to be the synaptotoxic species responsible for neurodegeneration in Alzheimer’s disease. Over the last 20 years, evidence has accumulated that implicates the N-terminus of Aβ as a region that may initiate the formation of damaging oligomeric species. Our laboratory has previously studied macrocyclic β-hairpin peptides derived from Aβ16–22 and Aβ30–36, capable of forming hexamers that can be observed by X-ray crystallography and SDS-PAGE. To better mimic oligomers of full length Aβ, we use an orthogonal protecting group strategy during the synthesis to append residues from Aβ1–14 to the parent macrocyclic β¬-hairpin peptide 1, which comprises Aβ16–22 and Aβ30–36. The N-terminally extended peptides N+1, N+2, N+4, N+6, N+8, N+10, N+12, and N+14 assemble to form dimers, trimers, and hexamers in solution-phase studies. X-ray crystallography reveals that peptide N+1 assembles to form a hexamer that is composed of dimers and trimers. These observations are consistent with a model in which the assembly of Aβ oligomers is driven by hydrogen bonding and hydrophobic packing of the residues from the central and C-terminal regions, with the N-terminus of Aβ accommodated by the oligomers as an unstructured tail. Chapter 3 describes the investigation of oligomers formed by macrocyclic β-hairpin peptides derived from Aβ12–40. Peptides 1a–i and 2a–d, derived from residues 12–40, demonstrate solution-phase and crystallographic assembly dependent on β-hairpin stability and intermolecular interactions mediated by N-terminal residues 12–14. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies reveal that peptides 1a and 1d assemble to form octamers, peptides 1h and 2a assemble to form octamers and tetramers, and peptide 2d assembles to form tetramers. X-ray crystallographic studies of peptide 2a reveals the assembly of a β-barrel-like tetramer stabilized by edge-to-edge hydrogen bonding and hydrophobic packing. Additional evidence for the assembly of a β-barrel-like octamer is also present within the crystal lattice. Replica-exchange molecular dynamics (REMD) simulations show that this tetramer can accommodate residues 23–29 as a loop and additional residues from the N- and C-terminal region of Aβ. Molecular dynamics simulations reveal that this REMD modeled tetramer can disrupt a lipid membrane and facilitate water permeation in a pore-like fashion. These observations provide novel insight into the mechanisms by which endogenous Aβ oligomers may assemble and damage neuronal membranes in the Alzheimer’s brain. Chapter 4 describes studies of macrocyclic β-hairpin peptides comprising β-strands of Aβ residues 17–22 and 30–36. The incorporation of an N-methyl group on position 20, in peptides derived from 17–22 and 30–36, alters the hydrogen bonding edges and assembly of triangular trimers formed by these peptides. To better mimic the hydrogen boding edges of β-hairpins formed by unmodified Aβ we investigated substitution of the N-methyl group on position 20 with an α-methyl group on position 19. The incorporation of α-methyl amino acids into macrocyclic β-hairpin peptides derived from presents an opportunity to study the structures of oligomers that may more closely resemble endogenous Aβ oligomers.