Abstract: To evaluate the role of Cys-X-Cys fragment in the formation of domains containing metal-thiolate cluster in metallothionein, we used site-directed mutagenesis to replace Asn-4 and Thr-27 in β-domain of monkey metallothionein Ⅰ with Cys. Thus, at the amino acids sequence level, the β-domain was designed to mimic the α-domain. Then we studied the recombinant monkey wild type metallothionein Ⅰ(WTmkMT), and mutants of mkMT N4C, mkMT T27C and mkMT N4C/T27C, using UVspectroscopy, CDspectroscopy, electrospray ionization mass spectroscopy, pHtitration and the reaction with DTNB [5,5′-dithiobis(2-nitrobenzoic acid)]. We found that in the mutants also existed a metal ion (Ⅱ)-thiolate cluster wrapped up by the peptides. In addition to the origin α-domain, a new domain containing 4-metal ion(Ⅱ)-thiolate cluster was formed in mkMT N4Cand mkMT N4C/T27C. And this new domain was more stable than the natural β-domain. The obtaining of mkMT N4C, mkMT T27Cand mkMT N4C/T27C laid down a base for the further study of the role of Cys-X-Cys fragment in the formation of metallothionein′s structure.

Key words: Monkey metallothionein Ⅰ, Mutants, ESI-MS