Herrmann H, Häner M, Brettel M, Ku N O, Aebi U
German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, D-69120, Germany.
J Mol Biol. 1999 Mar 12;286(5):1403-20. doi: 10.1006/jmbi.1999.2528.
We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.
我们已经确定了来自四个细胞质序列同源类别的重组中间丝（IF）蛋白不同早期组装产物的单位长度质量（MPL）组成，并将这些值与相应成熟丝的进行了比较。在标准组装条件下（即25 mM Tris-HCl（pH 7.5）、50 mM NaCl、37℃）孵育两秒后，波形蛋白、结蛋白和神经丝三联体蛋白NF-L聚集成相似类型的“单位长度丝”（ULF），而细胞角蛋白（CK）8/18在这个时间点已经形成了长的中间丝，因此必须降低离子强度。根据MPL值推断，每个丝横截面的分子数，CK8/18最低，即两秒时为16和25，而一小时时为16和21。NF-L的相应值为26和30。波形蛋白ULF表现出明显的异质性，两秒时主要峰值为32和45，一小时后为30、37和44。结蛋白形成的丝质量明显更高，组装两秒和一小时后每个横截面有47个分子。这表明不同类型的中间丝蛋白产生的丝每个横截面的分子数明显不同。此外，与相应的成熟中间丝相比，观察到的ULF表观丝直径显著减小是丝内“保守”的径向压缩型重组的结果，这是从以下事实得出的结论：未成熟丝和成熟丝每个横截面包含非常相似数量的亚基。此外，丝的MPL组成显著依赖于所采用的组装条件。例如，在0.7 mM磷酸盐（pH 7.5）、2.5 mM MgCl2中形成的波形蛋白纤维，与在标准条件下组装相比，每个横截面的分子数显著增加（56和84）。温度也强烈影响组装：高于某个阈值温度会形成“病理性”ULF并停滞在这种状态，这表明系统被迫在亚基之间形成强烈但无成效的相互作用。用发生突变的波形蛋白获得了类似的“死端”结构，这些突变在假定具有一般结构重要性的亚结构域中引入了主要改变，这表明这些序列变化导致了分子间相互作用的新模式。
