Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis.
A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.
Cols
N
N
Departament of Genètica, Facultat de Biologia, Universitat de Barcelona, Spain.
Romero-Isart
N
N
Capdevila
M
M
Oliva
B
B
Gonzàlez-Duarte
P
P
Gonzàlez-Duarte
R
R
Atrian
S
S
D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism*; D002411:Cation Exchange Resins; D002614:Chelating Agents; D002942:Circular Dichroism; D003001:Cloning, Molecular; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012117:Resins, Synthetic; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013499:Surface Properties; D015032:Zinc / Q000378:metabolism*
D002411:Cation Exchange Resins; C041582:Cd-Zn-metallothionein; D002614:Chelating Agents; D011994:Recombinant Proteins; D012117:Resins, Synthetic; C004203:cadmium-binding protein; C021267:zinc thionein; D002104:Cadmium; C024997:Chelex 100; D008668:Metallothionein; D015032:Zinc