Division of Molecular Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
J Struct Biol. 2012 Jan;177(1):54-62. doi: 10.1016/j.jsb.2011.11.003. Epub 2011 Nov 9.
We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8 M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025 g/l, unit-length filaments were abundantly present after 2s of assembly. During the following 5 min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length l(p) of K8/K18 filaments to be in the range of 300 nm. Addition of 1 mM MgCl(2) increases l(p) to 480 nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.
我们已经生成了人重组角蛋白 K8 和 K18，并描述了定量研究它们组装成纤维的条件。当从 8M 尿素中重新折叠到低离子强度/高 pH 缓冲液中时，K8 以二聚体到四聚体的形式存在，这可以通过分析超速离心法揭示。相比之下，K18 以单体形式沉淀。当在 8M 尿素中混合并一起重新折叠时，K8 和 K18 表现出与均相四聚体复合物相容的 s 值分布。这一发现通过沉降平衡离心得到了证实。随后，将这些四聚体起始单元在不同的蛋白质浓度下进行组装实验。在低浓度下（如 0.0025g/l），组装 2 秒后就会大量存在单位长度的纤维。在接下来的 5 分钟内，纤维迅速生长，通过测量单个纤维的长度，我们能够生成依赖时间的长度分布。这些数据表明，角蛋白 K8/K18 的组装速度比波形蛋白和结蛋白快几倍。此外，我们确定了 K8/K18 纤维的持久长度 l(p)在 300nm 范围内。添加 1mM MgCl2 会将 l(p)增加到 480nm，这表明镁离子在组装过程中在某种程度上影响了角蛋白亚基在纤维内的相互作用。
