Steinert P M
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Struct Biol. 1991 Oct;107(2):175-88. doi: 10.1016/1047-8477(91)90020-w.
The question as to whether keratin intermediate filaments (KIF) are built from a unique "building block" consisting of a pair of coiled-coil molecules has been studied by examining the earliest stages of reassembly of mouse K1/K10 KIF in vitro. Particles formed in protein solutions of about 45 micrograms/ml (near or below the critical concentration for assembly) or 0.5-1.65 mg/ml were monitored by turbidity, visualized by electron microscopy, and their structures resolved biochemically using crosslinking, limited proteolysis, and amino acid sequencing. The rate of KIF reassembly in vitro is limited by an initial slow step involving the formation of a three- or four-molecule oligomer. At 2 min, the particles in solution are about 65 nm long and consist of two molecules aligned antiparallel and staggered. A few minutes later, a three- and/or four-molecule species appears that may be the rate-limiting particle(s). It is also 65 nm long, but contains one or two additional molecules aligned in register but antiparallel with respect to one of the molecules on the two-molecule particle. The present data cannot establish whether the rate-limiting particle contains three or four molecules, or in fact consists of a mixture of both. Below the critical concentration for KIF assembly, it exists in solution in rapid exchange with particles containing one and two molecules. In solutions above the critical concentration for assembly, once this oligomer has formed in sufficient quantity, further assembly into KIF occurs rapidly; 90, 110, and 130-nm particles soon appear by apparent addition of a single molecule or oligomers containing two, three, four, or even several molecules. Within about 20 min short KIF about 200-500 nm long appear which later elongate to long (greater than 1 micron) KIF. These data suggest that KIF assembly requires the initial correct alignment of three or four molecules which, once formed, provides a template for further rapid addition of molecules leading to KIF assembly. Furthermore, the data establish that KIF are built from alternating rows of in-register and staggered antiparallel molecules. The present data confirm independently the observations of the previous paper and do not support earlier notions that IF are built from a tetrameric building block consisting of a pair of in-register molecules. Finally, the data suggest that the mechanism of assembly in vitro and the dynamic in vivo assembly-disassembly characteristics of KIF in particular and IF in general are mediated through a variety of small oligomeric species ranging in size from one to several molecules.
关于角蛋白中间丝（KIF）是否由一对卷曲螺旋分子组成的独特“构建块”构建而成这一问题，已通过在体外研究小鼠K1/K10 KIF重新组装的最早阶段进行了探讨。通过浊度监测在约45微克/毫升（接近或低于组装临界浓度）或0.5 - 1.65毫克/毫升的蛋白质溶液中形成的颗粒，用电子显微镜观察其形态，并使用交联、有限蛋白酶解和氨基酸测序对其结构进行生化解析。KIF在体外重新组装的速率受限于一个初始的缓慢步骤，该步骤涉及三分子或四分子寡聚体的形成。在2分钟时，溶液中的颗粒长约65纳米，由两个反平行且交错排列的分子组成。几分钟后，出现一种三分子和/或四分子的物种，它可能是限速颗粒。它同样长65纳米，但包含一个或两个额外的分子，这些分子与两分子颗粒上的一个分子对齐但反平行。目前的数据无法确定限速颗粒包含三个还是四个分子，或者实际上是否由两者的混合物组成。在低于KIF组装临界浓度时，它在溶液中与含有一分子和两分子的颗粒快速交换。在高于组装临界浓度的溶液中，一旦这种寡聚体形成足够数量，进一步组装成KIF的过程就会迅速发生；通过明显添加单个分子或含有两、三、四甚至几个分子的寡聚体，很快会出现90、110和130纳米的颗粒。在约20分钟内，出现约200 - 500纳米长的短KIF，随后它们会伸长成长（大于1微米）的KIF。这些数据表明，KIF组装需要三分子或四分子的初始正确排列。一旦形成，这为进一步快速添加分子以导致KIF组装提供了模板。此外，数据表明KIF由交替排列的对齐且交错的反平行分子行构建而成。目前的数据独立证实了前文的观察结果，并不支持早期关于中间丝由一对对齐分子组成的四聚体构建块构建而成的观点。最后，数据表明体外组装机制以及特别是KIF和一般中间丝在体内的动态组装 - 拆卸特性是通过从一分子到几分子不等的各种小寡聚体物种介导的。
