UC Irvine

The formation of aggregates of the amyloidogenic peptide β-amyloid (Aβ), termed oligomers, is central to Alzheimer’s disease, where oligomers play a central role in neurodegeneration. The use of chemical model systems can help provide insights into the structures and interactions of oligomers, which are otherwise difficult to study because they are heterogeneous and metastable. In my dissertation, I describe the application of new tools to study Aβ-derived oligomers to better understand the molecular basis of Alzheimer’s disease.Chapter 1 describes the use of macrocyclic β-hairpin peptides as model systems to study amyloid oligomers. The first part of the chapter describes the chemical synthesis of the macrocyclic β-hairpin peptides and covalent assemblies thereof. The second part of the chapter describes the characterization of the oligomers formed by the macrocyclic β-hairpin peptides, focusing on SDS-PAGE, size-exclusion chromatography (SEC), and X-ray crystallography. This chapter focuses on the β-amyloid peptide (Aβ), but these strategies are applicable to a broad range of amyloid-derived peptides and proteins. The subsequent chapters study the covalently stabilized trimers derived from residues 16–36 of Aβ as model systems to understand Aβ oligomers. Chapter 2 expands the toolset to study Aβ-derived oligomers using the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. Oligomer model 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues. Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues co-localize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells and establish the occurrence of FRET, suggesting intracellular oligomerization. Chapter 3 explores how differences in β-hairpin registration can affect biophysical and biological activity. The prevalence of β-sheet rich structures of Aβ, where the central and C-terminal regions fold to form a β-hairpin has been established as important secondary structure of Aβ oligomers. Using our laboratory’s template for oligomer models, two isomorphic trimers were synthesized and fluorescently labeled. The differences in the two trimers’ interactions with cells are explored through fluorescence microscopy. Results suggest that the alignment of the β-hairpin can affect cellular interactions and support the principle that function follows form. Chapter 4 introduces additional mass spectrometry techniques as tools in the investigation of the complex behavior of oligomer model systems. In particular, native MS is a powerful technique for the study of non-covalent assemblies that complements other biophysical techniques. In this study, the oligomeric assemblies of three isomorphic trimers are investigated by two native MS techniques, mass photometry, SDS-PAGE, and cellular cytotoxicity. These studies affirm the need for multiple techniques to accurately characterize oligomer behavior. Chapter 5 details techniques learned from my experience working in the UCI Mass Spectrometry Core Facility as the Mass Spectrometry Fellow. I have distilled the knowledge gathered through literature and experience into a simple guide that outlines the types of mass spectrometry instruments available in the UCI Mass Spectrometry Core Facility, sample preparation requirements and instrument compatibility, and data analysis resources. Finally, in my epilogue, I describe a selection of collaborative projects I have contributed to in my time in the Nowick laboratory. These collaborations include research related to amyloidogenic peptides, antibiotic peptides, and online organic chemistry teaching methods.