Molecular and biophysical characterization of assembly-starter units of human vimentin
Molecular and biophysical characterization of assembly-starter units of human vimentin
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DOI：
10.1016/j.jmb.2004.04.039
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发表时间：
2004-06-25
影响因子：
5.6
通讯作者：
Herrmann, H
中科院分区：
文献类型：
--
作者：
Mücke, N;Wedig, T;Herrmann, H
我们开发了一种基于磷酸盐缓冲系统的中间丝蛋白（IF）蛋白vimentin的组装方案，该方案能够动态形成真实的IF。这种生理缓冲液的优点是可以通过内部赖氨酸残基的化学交联来分析亚基相互作用。通过该系统，我们分析了线圈-线圈棒结构域之间的潜在相互作用，这些相互作用被认为对IF的形成和稳定性做出了重要贡献。我们发现，在低盐条件下二聚的无头波形蛋白在组装条件下结合成A（22）型构型的四聚体，这表明增加离子强度的影响之一是有利于线圈2-线圈2相互作用。此外，为了深入了解全长波形蛋白组装第一阶段发生的分子相互作用，我们采用了人类波形蛋白的温度敏感变体，它在室温下停留在“单位长度丝”（ULF）状态，但在温度升高到37度时开始拉长。最重要的是，我们通过交联分析证明，成熟IF中A（22）和A（12）交联产物的ULF形成表明，在IF组装的纵向退火和径向压实步骤中确实发生了主要的重排。(C) 2004 Elsevier Ltd.版权所有。
We have developed an assembly protocol for the intermediate filament (IF) protein vimentin based on a phosphate buffer system, which enables the dynamic formation of authentic IFs. The advantage of this physiological buffer is that analysis of the subunit interactions by chemical crosslinking of internal lysine residues becomes feasible. By this system, we have analyzed the potential interactions of the coiled-coil rod domains with one another, which are assumed to make a crucial contribution to IF formation and stability. We show that headless vimentin, which dimerizes under low salt conditions, associates into tetramers of the A(22)-type configuration under assembly conditions, indicating that one of the effects of increasing the ionic strength is to favor coil 2-coil 2 interactions. Furthermore, in order to obtain insight into the molecular interactions that occur during the first phase of assembly of full-length vimentin, we employed a temperature-sensitive variant of human vimentin, which is arrested at the "unit-length filament" (ULF) state at room temperature, but starts to elongate upon raising the temperature to 37 degreesC. Most importantly, we demonstrate by cross-linking analysis that ULF formation of A(22) and A(12) cross-linking products in mature IFs, however, indicates that major rearrangements do occur during the longitudinal annealing and radial compaction steps of IF assembly. (C) 2004 Elsevier Ltd. All rights reserved.