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(WT mkMT), and mutants of mkMT N4C, mkMT T27C and mkMT N4C/T27C, using UV spectroscopy, CD spectroscopy, electrospray ionization mass spectroscopy, pH titration 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 N4C and mkMT N4C/T27C. And this new domain was more stable than the natural \%β\%\|domain. The obtaining of mkMT N4C, mkMT T27C and 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.
The technique of coupling capillary liquid chromatography (CLC) with ESIMS was introduced in this article. The homemade reverse phase CLC was coupled with ESIMS to analyze the proteolysis of the BSA and TCS. Good separation efficiency is obtained in the peptide mapping and the partial sequence of peptides was deduced in the CID spectrum. Studies show that CLC-MS has high efficiency on sample separation, and has the merits of sensitivity, accuracy and speed on sample identification. CLC has the lower sample consuming and will be more widely used in the proteomics.
