Metalation and Structural Properties of Human Metallothionein-3

Amelia T. Yuan, The University of Western Ontario

Abstract

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.