The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression.
The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression.
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DOI：
10.1083/jcb.110.4.1199
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发表时间：
1990-04
期刊：
影响因子：
--
通讯作者：
Weber K
中科院分区：
文献类型：
--
作者：
Hatzfeld M;Weber K
重组 DNA 技术已用于分析角蛋白中间丝 (IF) 组装的第一步；即，形成双股螺旋线圈。对缺少半胱氨酸的角蛋白 8 和 18 进行位点特异性体外诱变，将两种角蛋白的 α 螺旋杆结构域中相同相对位置的一个氨基酸改变为半胱氨酸。突变位于七肽重复模式的“d”位置中杆的-36位，因此空气氧化可以引入零长度的胱氨酸交联。从大肠杆菌中单独纯化的突变角蛋白8和18很容易在2M盐酸胍中形成胱氨酸同二聚体，并且可以通过凝胶过滤与单体分离。当由两个还原单体形成的丝被氧化时，获得具有胱氨酸交联的异二聚体。随后在 8.5 M 尿素中进行的离子交换色谱显示仅形成了单一二聚体物质。对角线电泳和反相HPLC鉴定该二聚体为含有胱氨酸的异二聚体。这种异二聚体很容易再次组装成 IF，与从非突变对应物或真正的角蛋白获得的异二聚体无法区分。相反，胱氨酸稳定的同二聚体的混合物仅形成大的异常聚集体。然而，当添加还原剂时，丝再次形成并在氧化后产生异二聚体。因此，角蛋白 IF 组装中的必要杂聚物步骤似乎优先发生在二聚体水平，而不是在四聚体形成过程中。我们的结果还表明，角蛋白 I 和 II 同二聚体一旦形成，至少在 2 M 盐酸胍中是亚稳态物质，因为它们的混合物自发转化为异二聚体，除非同二聚体通过胱氨酸交联稳定。同型二聚体的这种先前意想不到的特性解释了有关角蛋白二聚体的文献中的主要差异。
Recombinant DNA technology has been used to analyze the first step in keratin intermediate filament (IF) assembly; i.e., the formation of the double stranded coiled coil. Keratins 8 and 18, lacking cysteine, were subjected to site specific in vitro mutagenesis to change one amino acid in the same relative position of the alpha-helical rod domain of both keratins to a cysteine. The mutations lie at position -36 of the rod in a "d" position of the heptad repeat pattern, and thus air oxidation can introduce a zero-length cystine cross-link. Mutant keratins 8 and 18 purified separately from Escherichia coli readily formed cystine homodimers in 2 M guanidine-HCl, and could be separated from the monomers by gel filtration. Heterodimers with a cystine cross- link were obtained when filaments formed by the two reduced monomers were allowed to oxidize. Subsequent ion exchange chromatography in 8.5 M urea showed that only a single dimer species had formed. Diagonal electrophoresis and reverse phase HPLC identified the dimer as the cystine containing heterodimer. This heterodimer readily assembled again into IF indistinguishable from those obtained from the nonmutant counterparts or from authentic keratins. In contrast, the mixture of cystine-stabilized homodimers formed only large aberrant aggregates. However, when a reducing agent was added, filaments formed again and yielded the heterodimer after oxidation. Thus, the obligatory heteropolymer step in keratin IF assembly seems to occur preferentially at the dimer level and not during tetramer formation. Our results also suggest that keratin I and II homodimers, once formed, are at least in 2 M guanidine-HCl a metastable species as their mixtures convert spontaneously into heterodimers unless the homodimers are stabilized by the cystine cross-link. This previously unexpected property of homodimers explains major discrepancies in the literature on the keratin dimer.