Abstract
Earthworms express, as most animals, metallothioneins (MTs)—small, cysteine-rich proteins that bind d10 metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.
Highlights
One of the most intriguing questions in the field of metal homeostasis concerns how biological systems distinguish and discriminate between different metal ions
As far as essential metal ions are concerned, it has been recognised that the cytosolic concentrations of essential metal ions are, in healthy conditions, regulated according to their relative position within the Irving-Williams series, and the proteins involved in their homeostasis typically have metal affinities to match these concentrations [1]
We have studied the composition, metal affinity, and folding behaviour of recombinant L. rubellus wMT-2 in its Zn(II)- and Cd(II)-bound forms
Summary
One of the most intriguing questions in the field of metal homeostasis concerns how biological systems distinguish and discriminate between different metal ions. This approach is in principle applicable to non-covalent protein complexes including metalloproteins in general, but has proven outstandingly useful for the study of MTs, as it is the only method that allows distinction between different metallospecies [41,42,43,44,45,46]. The significance of this observation will be discussed later on
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