Thesis Format

Integrated Article

Cu(I) Binding to Recombinant Metallothioneins

Author

Adyn Melenbacher, The University of Western OntarioFollow

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Stillman, Martin J.

Abstract

Copper is a vital metal in the human body. Due to copper redox chemistry, cellular Cu(I) concentrations must be carefully controlled through Cu(I) chaperone and storage proteins to avoid the production of reactive oxygen species (ROS). Metallothionein (MT) proteins bind Cu(I) and Zn(II) with high affinities in metal-thiolate clusters. While the stoichiometries of the metal-thiolate clusters forming after the addition of Cu(I) or Zn(II) to apo MT are well known, previous studies on the stoichiometries of the species that form when Cu(I) binds to Zn-MT have been limited by the methodologies used. In addition, whether the multiple isoforms of human MT have different metal binding properties is unclear. Furthermore, there is a lack of structural information on how the protein folds around Cu(I)-thiolate clusters. A novel electrospray ionization mass spectrometry (ESI-MS) method involving isotopically pure ⁶³Cu(I) and ⁶⁸Zn(II) was used to determine the Cu:Zn ratios in each Cu,Zn-MT species. Emission and circular dichroism (CD) spectroscopy were used to probe changes in the structure. The metal binding properties of the three most prevalent human MT isoforms were studied systematically so that their properties could be compared. NMR spectroscopy was used to directly investigate how the β domain of MT1A folds around a Cu₆S₉ cluster. The addition of ⁶³Cu(I) to ⁶⁸Zn₇-MT1A, ⁶⁸Zn₇-MT2, and ⁶⁸Zn₇-MT3 results in a series of Cu,Zn-MT species with distinct emission and CD spectral features. Similar species form in each isoform with the main species being Cu₅Zn₅-MT, Cu₆Zn₄-MT, Cu₉Zn₃-MT, and Cu₁₀Zn₂-MT. Analysis of the speciation resulting from Cu(I) addition to the apo or Zn₇ forms of MT1A, MT2, and MT3 reveals no significant optimization in Cu(I) binding for one isoform over the others. Preliminary NMR studies on Cd₃-β MT revealed that a stabilizing GB1 tag was necessary to reduce exchange processes impacting the NMR spectra. After optimization of the protein and parameters, the focus switched to Cu₆-GB1βMT, which was labelled with ¹³C and ¹⁵N. A series of three-dimensional NMR spectroscopy experiments were carried out and the chemical shifts of the C, N, and H atoms were assigned. Distance restraints were determined through NOESY experiments and preliminary protein structures were calculated.

The results discussed in this Thesis address the long-standing problems of determining exact metal ratios and structures of metallothionein proteins.

Summary for Lay Audience

Metals, such as zinc and copper, are necessary nutrients. Metal ion-dependent chemistry is used by many different enzymes to facilitate chemical reactions in humans. Due to this necessary chemistry, metal ions need to be chaperoned in the cell at all times to prevent unwanted reactions. This protection is carried out by essentially “hiding” the reactive metal ions in chaperone, transport, and storage proteins. The metallothionein (MT) protein is involved in the storage of Cu(I) and Zn(II). MT acts as a sponge and can bind many metal ions while forming different structures. There are four isoforms of the human MT protein that are all very similar in sequence. It is unknown whether these different isoforms have different metal binding properties. While the stoichiometry, or number of Cu(I) ions bound, is well known when starting with a metal-free MT protein, it is unclear what structures form when MT initially contains Zn(II). To study this, electrospray ionization mass spectrometry was used to determine the mass of the species forming after the addition of isotopically pure ⁶³Cu(I) to ⁶⁸Zn₇-MT. The use of a single isotope of Cu(I) and Zn(II) allowed for the number of Cu(I) and Zn(II) ions in each Cu,Zn-MT species to be precisely determined for the first time. To determine whether there were isoform-dependent differences, the three most prevalent MT isoforms were studied and compared systematically. Finally, the structure of the protein around the Cu(I) ions is not well known and so the fold of the β domain of MT1A around a Cu₆ cluster was investigated using nuclear magnetic resonance spectroscopy.

Recommended Citation

Melenbacher, Adyn, "Cu(I) Binding to Recombinant Metallothioneins" (2024). Electronic Thesis and Dissertation Repository. 10046.
https://ir.lib.uwo.ca/etd/10046

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