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Thesis Format

Integrated Article


Doctor of Philosophy




Stillman, Martin J.


Metallothioneins (MTs) are essential metalloproteins for humans. Mammalian MTs are 6-8 kDa in size and contain 20 conserved cysteines which bind metals. A typical MT protein can bind up to 7 divalent metals such as Zn(II) or Cd(II), and up to 20 monovalent metals such as Cu(I). The binding pathways of metals to MTs have been studied, however, semi-quantitative methods such as UV-visible absorption spectroscopy and the use of metal-sensors are not sufficient for studying the unique multi-metal binding pathways of MTs. Of interest is MT3, a brain-expressed MT with mostly qualitative and semi-quantitative reports of structure and metal-binding affinity. This is a significant gap in the literature, as MT3 is a growth inhibitory factor for neurons and a significant player in the mediation of neurodegenerative diseases.

This thesis presents a complete set of experiments defining the properties of MT3. Using methods such as electrospray ionization mass spectrometry, UV-visible absorption spectroscopy, ion mobility mass spectrometry, X-ray absorption spectroscopy, molecular modelling, and NMR spectroscopy, various aspects of metallothionein-3 have been explored, including: (i) the apo-MT3 structure, (ii) Cd(II) and Zn(II) metalation of MT3 as well as how this is impacted by carbonic anhydrase, (iii) the oxidation reactions of H2O2 with fully- and partially-metalated MT3, (iv) the reaction of Bi(III) with MT3, (v) the reaction of As(III) with MT3, (vi) the reaction of Hg(II) with MT3, and lastly, (vii), the supermetalation of MT3. The work in this thesis defines MT3 as a tightly compact apo-protein, with high and rapid reactivity with all metals and H2O2 in comparison to other MT isoforms. MT3 also forms a unique bridging thiolate structure with Bi(III) with distinct spectroscopic properties, as well as multiple coordination geometries with Hg(II). Lastly, MT3 can be supermetalated with Zn(II) and Cd(II), which can be characterized by 113Cd and 113Cd-1H HSQC NMR spectroscopy.

Summary for Lay Audience

Nutritional guidelines include inorganic minerals such as metals because they are used in various ways in the body. For example, zinc is used as a component of many processes including the communication between cells in the brain as well as the regulation of pH acidity and basicity in the cellular environment. In contrast, when a toxic metal such as arsenic is introduced, this metal can take the place of essential metals and disrupt finely tuned systems within the body. Metallochaperones, or proteins that bind to metals to donate them to the correct systems, can also be effective protectors from toxic metals. A metallochaperone of interest is that of the protein family called metallothionein, which are small proteins with high reactivity and capacity for metal binding. One of the isoforms of metallothionein is metallothionein-3, which is a protein that is known to be involved in diseases such as Alzheimer’s Disease and Parkinson’s Disease. It is mainly located in brain tissue in humans and has many unique properties, including a reported function of inhibiting the growth of neurons. This thesis outlines the properties of metallothionein-3, including how it reacts with many different metals, including essential and toxic metals. In addition, the work presented in this thesis focuses on the structural properties of metallothionein-3 and the larger implications this may have for the maintenance of health as well as sensitivity to disease. Some ways that disease states are studied include the introduction of reaction oxygen species, which are key components of any inflammatory responses as well as the introduction of stress factors into the physiological system. In summary, this work encompasses a broad survey of the ways in which metallothionein-3 can both regulate metals and how these metal-bound proteins can react with other additional factors such as an interacting protein or reactive oxygen species using precise analytical methods such as mass spectrometry.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Saturday, November 01, 2025