Date of Award
Doctor of Philosophy
Thanh D. Do
Bhavya Sharma, Ziling Xue, Rebecca Prosser
Ion mobility spectrometry – mass spectrometry (IMS-MS) is a powerful gas-phase technique that is routinely employed in the investigations of amyloid oligomers and conformational studies due to its ability to separate isobaric and isomeric species with the same mass-to-charge ratio (m/z). The goal of this dissertation is to use IMS-MS as a primary platform to probe the conformational landscape of a macrocyclic protein, Cyclosporin A (CycA), and to characterize interactions of amyloidogenic assemblies. Through five independent studies presented within document, the limits of IMS-MS are pushed by employing conditions which mimic biological environments.
Chapter 2 focused on how biologically relevant metal cations influence CycA structure. Ca2+ promoted three conformations of CycA which are mediated by cis-trans isomerization of the peptide backbone. The chameleonic effect exerted by Ca2+ helped explain mechanistic pathways which CycA utilizes within the body to bind to receptors.
Chapter 3 used phenol soluble modulin peptides to demonstrate the relevance of post-ion mobility dissociation, a phenomenon that can occur within IMS-MS instrumentation, in the interpretation of amyloid self-assembly data. A technique using post-IM dissociation was developed to discern differences among toxicity of the phenol soluble modulin peptides by observing their interactions with detergent micelles.
Chapters 4 and 5 are concerned with the molecular crosstalk of amyloid assemblies with species they can encounter in vivo. Studies of amylin and CGRP, two peptides that are cosecreted within the pancreas, showed that CGRP remodels amylin fibrillation. Furthermore, it is showed that amylin fibrillation is inhibited via formation of intermediary species upon complexation with Zn2+. Additionally, investigations of the interactions between Aβ [beta] and Tau showed that Aβ [beta] disrupted the formation of toxic disulfide-bonded Tau dimers.
Lastly, Chapter 6 is concerned with the feasibility of using small peptides, KV11 and its mutant G6W, as models for amyloid oligomers. Tissue viability assays showed that both peptides were able to replicate disease-like states which rival what is seen in neurodegenerative disease within mice brain tissue slices. Furthermore, IMS-MS and X-ray crystallography showed that G6W can assemble into a unique dodecameric assembly which can promote the release of disease-related neuropeptides from acute tissue slices.
Gray, Amber LeAnn Hope, "Elucidating molecular structure and interactions of disease-related noncovalent assemblies through ion mobility spectrometry - mass spectrometry. " PhD diss., University of Tennessee, 2022.