Construction of alpha-alpha domains structure in recombinant monkey metallothionein-1.
The differences in metal-thiolate coordination and reactivity of mammalian metallothionein (MT) domains are closely related to their distinct, highly conservative cysteine number and position. Monkey metallothionein-1, containing a beta-domain with Cd(3)S(9) cluster and an alpha-domain with Cd(4)S(11) cluster, was used to evaluate the role of cysteine residues in the formation of MT's metal-thiolate clusters. The possible influence of cysteine residues on the binding and stability of MT domains has been examined with the metallothionein mutants: N4C, T27C and N4C/T27C, which possess ten or eleven cysteine residues in the re-constructed beta-domain, respectively. Assisted by study of UV, CD and electrospray ionization mass spectroscopy (ESI-MS) and their reactivity with DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), we found that besides the original alpha-domain, some kinds of new domain containing 4-cadmium-thiolate clusters were formed in the N4C and N4C/T27C mutants of mkMT1. These new domains displayed metal binding and kinetic reactivity with DTNB similar to the alpha-domain. However, the thermal stability of the mutants was less stable than that of WT mkMT1. This might result from the disturbance of the inter-domains hydrogen bonds and of the non-cysteine amino acid residue arrangement.
Huang
Zhong Xian
ZX
Chemical Biology Lab, Department of Chemistry, Fudan University, 200433, Shanghai, China. zxhuang@fudan.edu.cn
Gao
Yuan
Y
Yu
Wen Hao
WH
Zhang
Su Yan
SY
Yang
Peng Yuan
PY
D000818:Animals; D002104:Cadmium / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D000882:Haplorhini; D006863:Hydrogen-Ion Concentration; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein; D004228:Dithionitrobenzoic Acid; D003545:Cysteine