TY - JOUR
T1 - Earthworm Lumbricus rubellus MT-2
T2 - Metal binding and protein folding of a true cadmium-MT
AU - Kowald, Gregory R.
AU - Sturzenbaum, Stephen
AU - Blindauer, Claudia A.
PY - 2016/1/5
Y1 - 2016/1/5
N2 - 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 by3H 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.
AB - 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 by3H 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.
KW - Cadmium
KW - Mass spectrometry
KW - Metallothionein
KW - NMR spectroscopy
KW - Protein folding
KW - Selectivity
KW - Zinc
UR - http://www.scopus.com/inward/record.url?scp=84954427790&partnerID=8YFLogxK
U2 - 10.3390/ijms17010065
DO - 10.3390/ijms17010065
M3 - Article
AN - SCOPUS:84954427790
SN - 1661-6596
VL - 17
SP - 1
EP - 16
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 1
M1 - A57
ER -