Hatzfeld M, Weber K
Max Planck Institute for Biophysical Chemistry, Goettingen, Federal Republic of Germany.
J Cell Biol. 1990 Apr;110(4):1199-210. doi: 10.1083/jcb.110.4.1199.
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.
重组DNA技术已被用于分析角蛋白中间丝（IF）组装的第一步，即双链卷曲螺旋的形成。缺乏半胱氨酸的角蛋白8和18经过位点特异性体外诱变，将两种角蛋白α-螺旋杆状结构域相同相对位置的一个氨基酸替换为半胱氨酸。这些突变位于杆状结构的-36位，处于七肽重复模式的“d”位置，因此空气氧化可引入零长度的胱氨酸交联。分别从大肠杆菌中纯化得到的突变角蛋白8和18在2M盐酸胍中很容易形成胱氨酸同型二聚体，并且可以通过凝胶过滤与单体分离。当由两种还原单体形成的细丝被氧化时，可获得具有胱氨酸交联的异型二聚体。随后在8.5M尿素中进行离子交换色谱分析表明，只形成了一种二聚体。对角线电泳和反相高效液相色谱鉴定该二聚体为含胱氨酸的异型二聚体。这种异型二聚体很容易再次组装成与非突变对应物或天然角蛋白获得的中间丝无法区分的中间丝。相比之下，胱氨酸稳定的同型二聚体混合物仅形成大的异常聚集体。然而，当加入还原剂时，细丝会再次形成，并在氧化后产生异型二聚体。因此，角蛋白IF组装中必不可少的异源多聚体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同型二聚体一旦形成，至少在2M盐酸胍中是亚稳物种，因为它们的混合物会自发转化为异型二聚体，除非同型二聚体通过胱氨酸交联得到稳定。同型二聚体这种先前未预料到的性质解释了文献中关于角蛋白二聚体的主要差异。
