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
本研究旨在探究不同金属离子(NaCl、KCl、CaCl_(2)、AlCl_(3)和FeCl_(3))对乳清分离蛋白(whey protein isolate,WPI)纤维聚合物聚合动力学、形态和结构的影响。结果表明,不同金属离子影响了WPI纤维聚合物的最终结构和特征。NaCl、KCl和C...本研究旨在探究不同金属离子(NaCl、KCl、CaCl_(2)、AlCl_(3)和FeCl_(3))对乳清分离蛋白(whey protein isolate,WPI)纤维聚合物聚合动力学、形态和结构的影响。结果表明,不同金属离子影响了WPI纤维聚合物的最终结构和特征。NaCl、KCl和CaCl_(2)对WPI的聚集影响较小,增加了溶液pH和电导率,而AlCl_(3)和FeCl_(3)加速了WPI的聚集,降低了溶液pH,增加了溶液电导率;与原始WPI纤维聚合物硫黄素T(Th T)荧光强度(388.92)相比,NaCl、KCl和CaCl_(2)增加Th T荧光强度分别至465.39、433.37和486.83,增加了WPI纤维聚合物的生成量,AlCl_(3)和FeCl_(3)降低Th T荧光强度分别至228.81和90.24,降低了WPI纤维聚合物的生成量;不同金属离子均改变了WPI聚合动力学,降低了(df/dt)_(max)值,并改变了滞后时间;NaCl、KCl和CaCl_(2)对WPI纤维聚合物的形态影响较小,AlCl_(3)和FeCl_(3)使WPI纤维聚合物更团簇、粗而杂乱;傅里叶红外光谱结果表明,NaCl、KCl和CaCl_(2)促进β-折叠结构生成,而AlCl_(3)和FeCl_(3)抑制了β-折叠生成,不同金属离子的加入均改变了纤维中β-链间的特征距离。这些结果为利用WPI制备不同聚集形态的纤维聚合物提供了科学依据。展开更多
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.
