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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Richard W. Vachet
Analytical Chemistry | Biochemistry | Chemistry
Amyloid-forming proteins are implicated in a number of debilitating diseases. While many amyloid-forming proteins are well studied, the early stages of amyloidosis are still not well understood on a molecular level. Covalent labeling, combined with mass spectrometry (CL-MS), is uniquely well suited to provide molecular-level insight into the factors governing the early stages of amyloidosis. This dissertation leverages CL-MS techniques to examine the early stages of β-2-microglobulin (β2m) amyloidosis. β2m is the protein that forms amyloids in the condition known as dialysis-related amyloidosis. An automated CL-MS technique that uses dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide as a labeling reagent was developed and used to measure energy barriers to the initial pre-amyloid structural change of β2m under different amyloid-forming conditions. The results represent the first ever measure of the activation barrier for a structural change initiating amyloid formation. The results also give new mechanistic insight into β2m’s amyloidogenic structural change, particularly the role of Pro32 isomerization. The catalytic nature of Cu(II) as an initiator of the β2m pre-amyloid structural change was confirmed as it significantly lowered the energy barrier to this structural change. It also appears that, when initiated by acid, the Pro32 isomerization may no longer be the rate limiting step in this process.
The same technique was further used to investigate the β2m structural change caused by its interaction with an amyloidogenic variant of β2m called ΔN6-β2m, which is missing its first six N-terminal residues. Both primary and secondary nucleation events involved in the β2m/ΔN6-β2m interaction were investigated. The measured barrier for the primary nucleation event seems to indicate that, like the acid induced structural change of β2m, the isomerization of Pro32 may not be the rate determining step. However, the measured barrier for the secondary nucleation event, similar to that of the Cu(II) induced structural change, indicates that the isomerization of Pro32 is the rate determining step. The use of point mutants gives further detail on the crucial residues and regions of the proteins required for a productive interaction to yield amyloids. The new kinetic and thermodynamic information gained in this work yields new insight into the mechanistic details of the biomedically important process of β2m amyloidosis.
Arden, Blaise G., "Amyloidogenesis of β-2-microglobulin Studied by Mass Spectrometry and Covalent Labeling" (2021). Doctoral Dissertations. 2264.