Doctor of Philosophy
Dr. Martin J. Stillman
Metallothioneins (MT) are a family of small cysteine rich proteins, which have been implicated in toxic metal detoxification, protection against oxidative stress, and as a metallochaperone. The most well studied member of the family is the mammalian MT, which consists of two domains: a β-domain with 9 cysteine residues, which sequesters 3 Cd2+/Zn2+, and an α-domain with 11 cysteine residues, which sequesters 4 Cd2+/Zn2+. The exact functions of MT are unknown but must relate to its metalation status. Several areas that could lead to the assignment of function include 1) the determination of the exact mechanism of metalation and the structural characterization of 2) submetalated and 3) supermetalated forms of MT.
The following thesis presents electrospray ionization mass spectrometric (ESI MS) data showing that the mechanism of metalation of MT is noncooperative. That is metalation events occur independently of each other, allowing for partially metalated species to exist in vivo. Further metalation studies using the isolated domains of MT as metal ion competitors against the full MT protein have yielded evidence that a new Zn5-MT exists in which both domains ‘coalesce.’ In addition, NMR and CD spectroscopy, have shown the existence of a new ‘supermetalated’ Cd8-MT, which also results in a ‘coalescence’ of both domains. Taken together these results indicate that 1) partially metalated forms of the protein are stable and 2) the traditional structure of MT is in fact the exceptional case and that under conditions of metal ion deficiency and excess, both domains interact with each other.
Sutherland, Duncan E K, "Structural Motifs of Novel Metallothionein Proteins" (2012). Electronic Thesis and Dissertation Repository. 489.