The fluorescence quenching spectra of the holo-Cu A and apo-Cu A domain of cytochrome c oxidase from Para coccus versutus by KI were investigated.The results indicated that in solution part of the fluorophore tryptoph...The fluorescence quenching spectra of the holo-Cu A and apo-Cu A domain of cytochrome c oxidase from Para coccus versutus by KI were investigated.The results indicated that in solution part of the fluorophore tryptophanes of the Cu A domain are exposed to the surface of the protein,and others buried inside o f the hydrophobic core.The fluorescence quenching constants of holo-Cu A and apo-Cu A domain protein by KI are1.39and2.24,respectively,which suggested t hat the bi-nuclear copper center[Cu A+1.5 -Cu A+1.5 ]also plays a role on the structural stability of the protein.展开更多
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...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.展开更多
文摘The fluorescence quenching spectra of the holo-Cu A and apo-Cu A domain of cytochrome c oxidase from Para coccus versutus by KI were investigated.The results indicated that in solution part of the fluorophore tryptophanes of the Cu A domain are exposed to the surface of the protein,and others buried inside o f the hydrophobic core.The fluorescence quenching constants of holo-Cu A and apo-Cu A domain protein by KI are1.39and2.24,respectively,which suggested t hat the bi-nuclear copper center[Cu A+1.5 -Cu A+1.5 ]also plays a role on the structural stability of the protein.
文摘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.
