Structure and properties of earthworm metallothionein-2

Since their discovery in the 1950s, metallothioneins (MTs) have been isolated from an array of organisms, from sea urchins to sheep, and pigs to peas. With the advent of nuclear Overhauser effect nuclear magnetic resonance (NMR) experiments, a powerful route for MT structure determination became available. The contribution of structural data obtained by NMR has enabled identification of hitherto unknown metallothionein motifs, thought to be significant in the ‘tuning’ of MT for specific roles or functions. As the MT superfamily is so broad, there is a wealth of information still to be uncovered for MTs from some of the lesser-studied organisms. Although many in vivo genomics-based investigations have been performed within the invertebrate family, proteomics-based investigations, especially with respect to terrestrial invertebrates, are sparse. This research has investigated the metal-binding behaviour and determined the solution structure of earthworm metallothionein-2 (wMT-2) from Lumbricus rubellus. In addition, a method for purifying both wMTs expressed in adult earthworms is presented; in contrast to previous studies the purification method includes the removal of the Nterminal S●tag. Using modified purification methods, the novel protein wMT-3 was also able to be isolated and characterised. Electrospray-ionisation mass spectrometry (ESIMS) revealed that all three wMTs bind 7 equivalents of divalent metal ions as the major species at pH ≈ 8. An NMR investigation of cleaved Cd-wMT-2 indicated that metalbinding is localised in two discrete clusters. Conversely to vertebrate MTs, but similar to some invertebrate MTs, the stoichiometry of the domains is of an M4Cys11- N-terminal cluster, followed by an M3Cys9- cluster. When challenged with protons, demetallation of cleaved Cd-wMT-2 occurred in a step-wise manner: a 3 metal loss being followed by a 4 metal loss; behaviour which is unique to the cadmium form. A comparison of the NMR TOCSY spectra of cleaved Cd- and Zn-wMT-2 showed significant differences, indicating that wMT-2 has a strong metal-binding preference for cadmium in vitro. In conclusion, the studies performed within this thesis have enabled the calculation of the first 3-dimensional solution structure of an MT from a terrestrial invertebrate, wMT-2. In addition, the novel protein wMT-3 was successfully isolated and purified