Doctor of Philosophy
Dr. Martin J. Stillman
More than 25% of proteins require metal ion cofactors for structure or function. The interactions between metalloproteins have largely been overlooked, though these interactions ultimately govern metal localization and control metal ion homeostasis. Mammalian metallothionein (MT) is a small, cysteine-rich metalloprotein that binds numerous metal ions per protein strand. Up to seven divalent metals, such as zinc or cadmium, are wrapped into a clustered two-domain structure. This unusually high metal content places MT as an attractive candidate for studying interactions with other metal-binding proteins. This present study investigates the metal transfer reactions between MTs and other metalloproteins, using carbonic anhydrase (CA) as a putative zinc-dependent enzyme.
This thesis presents electrospray ionization mass spectrometric (ESI-MS) data showing the competitive zinc metallation reactions between apoCA and various apoMTs. Modelling of the ESI-MS data is used to determine the reaction parameters and those parameters are shown to be reflected directly in the raw data. These results demonstrate how MT can act as a homeostatic buffer of metal ions, by binding them with different affinities. The kinetics of the metal transfers between zinc MTs and cadmium or zinc CA show that the rates of metal transfer between the two metalloproteins is directly dependent on the metal content of the MT. Further studies on the domain specific properties of MT using shortened MT domain fragment proteins show that: (i) there is no significant degree of domain specificity in metal binding to apoMTs; (ii) the weakest bound metal ion is located within the N-terminal domain of the intact MT protein; (iii) the highest affinity binding site is located within the C-terminal domain; and, (iv) domain-domain interactions within the MT peptide strand modulate metal binding affinities. Taken together, these results support the homeostatic roles of metallothionein proteins while also challenging the mechanisms for metal binding and release to apoenzymes.
Pinter, Tyler B. J., "Reactions between Zinc Metallothionein and Carbonic Anhydrase" (2015). Electronic Thesis and Dissertation Repository. 3212.