Date of Award

5-2024

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Marianna Torok

Second Advisor

Bela Torok

Third Advisor

Michelle Foster

Abstract

Amyloids are highly insoluble protein aggregates with fibrillar morphology and common physicochemical properties. The accumulation of toxic amyloids often leads to cell death or failure to function adequately; thus, they are associated with many human diseases termed amyloidosis. They are particularly prevalent in neurodegenerative disorders including Alzheimer’s disease but are also implicated in many systemic diseases such as type II diabetes, and cancer with increased impact on the aging population. In contrast to these toxic misfolded proteins, amyloids have also been shown to have specific biological functions within the human body. These functional amyloids exist in highly regulated environments and further understanding of the parameters surrounding their control will contribute to development of new therapeutic opportunities. Insulin is often used as a model amyloid and additionally exhibits cytotoxic effects making it an excellent peptide for amyloid studies. Calcium and zinc both contribute to the stability of insulin; however, metal cation dyshomeostasis has been linked to a variety of diseases including those linked to toxic amyloids. In this study, the effects of the Ca2+ and Zn2+ concentrations on insulin aggregation kinetics and morphology were investigated under acidic conditions using thioflavin T fluorescence spectroscopy, atomic force microscopy and Raman spectroscopy. Ca2+ exhibits an accelerating effect on fibril formation and generates polymorphic fibrils within a single sample. Zn2+ has an inhibitory effect on insulin fibril formation at concentrations of 25 mM or higher once the peptide reaches the elongation phase of aggregation. Cation identity altered insulin amyloid morphology yielding differing Raman signal for fibrils formed in the presence of Zn2+ or Ca2+ indicating distinct secondary structure of the protein. Unique branched fibrils and elongation kinetics were observed for samples containing Ca2+ or Zn2+ suggesting that these metal ions can alter the aggregation pathway. These results contribute to the current scientific knowledge on amyloid species to advance our understanding of these complex protein structures.

Comments

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