Arsenic metalation studies of metallothionein

Metalloproteins represent thirty percent of all reported proteins. One of the most well-studied metalloproteins is metallothionein. Mammalian metallothionein is a small, cysteine-rich, two-domain multiple-metal-binding protein. When bound to divalent metals (M Cd(II) or Zn(II)), mammalian metallothionein forms M3Cys9 (beta) and M4Cys11 (alpha) metal-thiolate clusters in two domains. Metalation of metal-free metallothionein is an example of metal-induced folding. Metal-induced folding occurs when protein folding is induced or dependent on the presence or type of one or more metals. This process is different from metal insertion into an already folded apo-protein. This present study investigates the metalation of metallothionein and the metal-induced folding pathway mechanism for metallothionein using electrospray ionization mass spectrometry to probe metal binding.;This thesis presents mass spectrometry data showing the arsenic-metalation of seaweed Fucus vesiculosus metallothionein, human metallothionein and its corresponding recombinant fragments. The results reported include: (1) the rate constants and transition state parameters for the arsenic-metalation of human and seaweed metallothionein; (2) the mechanism of arsenic-metalation is noncooperative; (3) the rate constant for the first incoming metal is dependent on the number of equivalent binding sites; (4) evidence of interdomain metal coordination; (5) evidence for the importance of the interdomain linker and domain specificity for arsenic-metalation; and (6) characterization of the novel metallothionein from earthworm Lumbricus rubellus.;Keywords. Metallothionein, arsenic, metalation pathways, kinetics, metal-induced folding, ESI mass spectrometry, Fucus vesiculosus, Lumbricus rubellus, Eyring transition state parameters, metal-binding mechanisms.