Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Martin Stillman


Metals are required by a quarter of all proteins to achieve their biological function, whether in an active site involved in catalytic chemistry or in a structural capacity. Metals are tightly regulated at the cellular level due to their propensity to cause unwanted side reactions and to be scavenged for use by pathogens. One of the proteins involved in this regulation of metal homeostasis is metallothionein (MT) which is a small, cysteine rich protein primarily involved in the regulation of zinc and copper homeostasis and heavy metal detoxification. MT is unique in its high cysteine content (~30% of the residues), its high capacity for metal binding and its fluxional structure in the absence of metal saturation. This fluxionality has made the structure of apo- and partially-metalated MTs difficult to study and as a result the binding pathway of MT for various metals remains unclear.

This thesis describes the hard-to-characterize structure of apo- and partially-metalated MTs, their binding pathways and potential applications. Using primarily electrospray ionization mass spectrometry (ESI-MS) and covalent labeling, the structure of apo- and partially metalated MTs was probed. Modeling techniques that generate simulated ESI-MS data were used to recreate the covalent labeling spectra and aid in the interpretation of this complicated reaction. These experiments showed that apo-MT adopts a compact, globular conformation that is resistant to initial modification by alkylating reagents. Furthermore, this compact conformation is essential to the fast kinetics of cadmium binding and cluster formation. This cluster formation was found to be pH dependent and this insight was essential in the design of an MT-based biosensor for the detection of As(III) and Hg(II). Altogether, these results reconcile previous conflicting reports about the metal binding mechanisms of MTs, provide evidence of compact conformations of apo-MT and its role in binding kinetics and begin to demonstrate potential application of this fundamental knowledge in the design and testing of an electrochemical MT-based biosensor.