Imam, Hasan T. (2015) Understanding cluster topology and metal-cluster dynamics of two zinc binding plant metallothionein isoforms. PhD thesis, University of Warwick.

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Abstract

Metallothioneins (MTs) are small metalloproteins, ubiquitously found in every phylum. Type 4 MTs (MT4s) from plants are attracting significant attention because of their frequent occurrence in plant embryos. The proposed function of MT4s is to store and distribute Zn2+ ions during seed germination. It is thus plausible that manipulation of MT4s could alleviate Zn2+ deficiency in cereal products. A better understanding of structure and metal binding dynamics of MT4s is necessary to use them as a tool to maintain seed Zn2+ nutritional value and to improve crop production. The dicotyledonous plant Arabidopsis thaliana has two seed-specific MT4s isoforms, MT4a and MT4b. The proteins have been expressed in E. coli and purified proteins have been found to bind 6 Zn2+ ions, like their homolog from wheat EC. For wheat EC, 6 Zn2+ ions bind in two domains - domain I binds 2 Zn2+ ions to form a binuclear cluster (Zn2Cys6); for domain II, a mononuclear (ZnHis2Cys2) site and a 6 membered ring cluster (Zn3Cys9) has been proposed. In Cd2+ saturated protein, domain II was unfolded, rendering study of metal-ligand connectivities by means of 111/113Cd NMR spectroscopy impossible. It has also been shown that the mononuclear site plays a significant role in proper folding of domain II, but only in presence of Zn2+ but not Cd2+. The focus of this research is to extend knowledge and understanding of the cluster topology, particularly regarding that of the domain II 3-metal cluster, and metal binding dynamics of MT4a and MT4b, using techniques including UV-Visible spectroscopy, native Electrospray Ionization-Mass Spectrometry (ESI-MS) and solution Nuclear Magnetic Resonance (NMR) spectroscopy.

Like wheat EC, domain II of MT4a and MT4b was misfolded when Cd2+ saturated. A possible solution to the absence of 1H and 111Cd resonances in fully Cd2+- substituted MT4s has been developed, based on mixed Zn/Cd MT4 species, which would contain Zn2+ in the isolated His2Cys2 site to achieve ordered protein folding, with most of the other sites occupied by NMR-active 111Cd2+. For mixed metal species (Zn/Cd) at 4 equivalents of Cd2+, a maximal number of metal-toligand connectivities was observed, allowing some new insights into the topology of the 3-metal cluster of MT4s. Metal transfer reactions with EDTA suggested that the 3 metals in this cluster were kinetically not equivalent, as one labile metal site within this domain was identified. Combination of connectivities data and metal transfer dynamics results allowed building an alternative 3 metal cluster – a binuclear M2Cys6 cluster connected to a single metal ion through one bridging cysteine.

Despite 84% sequence identity, MT4a and MT4b have shown remarkable differences in their metal binding dynamics against metal chelators, oxidants, protons, and phytate. Although both isoforms have very similar pH of half dissociation values, proton-induced Zn2+ release from MT4a was found to be cooperative while from MT4b, it was non-cooperative. MT4a transferred Zn2+ faster than MT4b to the metal chelators ethylenediaminetetraacetic acid (EDTA) and 4-(2-pyridylazo)resorcinol (PAR), and released Zn2+ faster during oxidation by the disulfide reagent 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB). While MT4b was prone to oxidation by the reactive oxygen species hydrogen peroxide (H2O2), MT4a resisted oxidation elicited by H2O2. The anti-nutrient phytate alone had very little effect on Zn2+ mobilisation from both MT4s but it accelerated Zn2+ transfer to PAR from both isoforms; however, the effect was more pronounced for MT4a than for MT4b. Glutathione disulfide (GSSG) and the glutathione redox couple (GSH/GSSG) were found to release Zn2+ from MT4b. Moreover, GSSG was found to form a non-covalent adduct with MT4b. Both isoforms exhibited domain-specific dynamics, where domain II was often more prone to metal transfer or release than domain I.

The findings of this research have enhanced the knowledge and understanding of cluster topology and metal-binding dynamics of MT4s, particularly MT4a and MT4b from Arabidopsis thaliana.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QD Chemistry
Official Date:	2015
Dates:	Date Event 2015 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Blindauer, Claudia
Extent:	xvi, 230 leaves : illustrations (some colour), charts
Language:	eng

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