Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Stillman, Martin J.

Abstract

Metallothioneins (MT) are a family of cysteine-rich metal-binding intrinsically disordered proteins that are ubiquitous across life. The proposed functions of MTs are to maintain Zn(II) homeostasis, participate in heavy metal detoxification, and protect against oxidative stress by binding to a variety of metals in a range of stoichiometries. However, due to their disordered nature and ability to form multiple metallated species, these diverse structures are not well characterized. Electrospray ionization mass spectrometry (ESI-MS) is a technique that allows quantification of heterogeneous metallation states through analysis of the speciation distributions. In this thesis, ESI-MS in combination with other spectroscopic techniques is used to probe the structural features of apo-MT, the early Zn(II) and Cd(II) metallation steps, the binding of the xenobiotic metal Bi(III), and the pathway of oxidation and subsequent Zn(II) and Cd(II) displacement upon exposure to H2O2. These speciation details shown using ESI-MS have not yet been described using other techniques. It was concluded that both the apo-MT structure and the metal-loading status impact MT’s metallation and oxidation properties, which provides insight into the structure-function relationship of MTs.

Summary for Lay Audience

Many proteins require metals such as zinc or copper for their physiological functions. However, the correct balance of these metals must be maintained as a concentration that is too low or too high may be toxic to cells. Additionally, environmental exposure to toxic heavy metals can lead to adverse health effects. Metallothioneins (MTs) are cysteine-rich, low molecular weight proteins that bind a variety of metals, balancing the availability of nutritionally essential metals and providing protection against toxic heavy metals. MTs may also act as an antioxidant. However, this process results in the release of bound metals from MT. The pathways of metal binding and metal release are relevant to our understanding of how MTs function, but these pathways are not currently well-described. MTs can bind multiple metals at once, making determination of the individual states difficult. Mass spectrometry (MS) is a technique that can identify these individual species and their relative concentrations based on mass changes caused by the binding of each metal. Using this technique, the metal binding pathways and metal release pathways to and from MTs can be described. The effect of the MT structure on these processes can also be considered, providing more insight into the relationship between protein structure and function.

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