Cody C W, Huang P C
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205-2103.
Biochem Biophys Res Commun. 1994 Jul 29;202(2):954-9. doi: 10.1006/bbrc.1994.2022.
Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, including three in each of two metal binding domains. We used site-directed mutagenesis to replace these intradomain lysines in Chinese hamster ovary MT2 with glutamic acid and/or glutamine. These mutant MTs were expressed in a metal sensitive yeast host. One mutant which had all three lysines in the alpha-domain replaced by glutamates (K43,51,56E) exhibited a reduced ability, relative to native MT, to protect yeast transformants against otherwise toxic levels of cadmium. This triply substituted mutant also exhibited anomalous migration on a non-denaturing gel relative to wild type MT and other MT lysine mutants, suggesting that the intradomain lysines are important in maintaining the conformational integrity of MT.
哺乳动物金属硫蛋白（MTs）有八个高度保守的赖氨酸残基，其中两个金属结合结构域各有三个。我们利用定点诱变技术，将中国仓鼠卵巢MT2中这些结构域内的赖氨酸替换为谷氨酸和/或谷氨酰胺。这些突变型MTs在对金属敏感的酵母宿主中表达。其中一个突变体，其α结构域中的所有三个赖氨酸都被谷氨酸替代（K43、51、56E），相对于天然MT，该突变体保护酵母转化体免受镉毒性水平影响的能力有所降低。相对于野生型MT和其他MT赖氨酸突变体，这个三重取代的突变体在非变性凝胶上也表现出异常迁移，这表明结构域内的赖氨酸对于维持MT的构象完整性很重要。
