Kirmse Robert, Portet Stephanie, Mücke Norbert, Aebi Ueli, Herrmann Harald, Langowski Jörg
Division of Biophysics of Macromolecules, German Cancer Research Center, Im Neuenheimer Feld 580, Heidelberg D-69120, Germany.
Department of Mathematics, University of Manitoba, Winnipeg MB, Canada R3T 2N2.
J Biol Chem. 2007 Jun 22;282(25):18563-18572. doi: 10.1074/jbc.M701063200. Epub 2007 Apr 2.
In vitro assembly of intermediate filament proteins is a very rapid process. It starts without significant delay by lateral association of tetramer complexes into unit-length filaments (ULFs) after raising the ionic strength from low salt to physiological conditions (100 mM KCl). We employed electron and scanning force microscopy complemented by mathematical modeling to investigate the kinetics of in vitro assembly of human recombinant vimentin. From the average length distributions of the resulting filaments measured at increasing assembly times we simulated filament assembly and estimated specific reaction rate parameters. We modeled eight different potential pathways for vimentin filament elongation. Comparing the numerical with the experimental data we conclude that a two-step mechanism involving rapid formation of ULFs followed by ULF and filament annealing is the most robust scenario for vimentin assembly. These findings agree with the first two steps of the previously proposed three-step assembly model (Herrmann, H., and Aebi, U. (1998) Curr. Opin. Struct. Biol. 8, 177-185). In particular, our modeling clearly demonstrates that end-to-end annealing of ULFs and filaments is obligatory for forming long filaments, whereas tetramer addition to filament ends does not contribute significantly to filament elongation.
中间丝蛋白的体外组装是一个非常快速的过程。在将离子强度从低盐提高到生理条件（100 mM KCl）后，四聚体复合物通过横向缔合形成单位长度丝（ULF），组装过程几乎没有明显延迟就开始了。我们采用电子显微镜和扫描力显微镜，并辅以数学建模，来研究人重组波形蛋白的体外组装动力学。根据在增加组装时间时所测量的所得丝的平均长度分布，我们模拟了丝的组装并估计了特定反应速率参数。我们为波形蛋白丝的伸长建立了八种不同的潜在途径模型。将数值模拟结果与实验数据进行比较后，我们得出结论，涉及ULF快速形成，随后是ULF和丝退火的两步机制是波形蛋白组装最可靠的情况。这些发现与先前提出的三步组装模型的前两步一致（Herrmann, H., and Aebi, U. (1998) Curr. Opin. Struct. Biol. 8, 177 - 185）。特别是，我们的建模清楚地表明，ULF和丝的端对端退火对于形成长丝是必不可少的，而在丝末端添加四聚体对丝的伸长贡献不大。
