Polymerisation of chemically cross-linked actin:thymosin β4 complex to filamentous actin:: Alteration in helical parameters and visualisation of thymosin β4 binding on F-actin
Polymerisation of chemically cross-linked actin:thymosin β4 complex to filamentous actin:: Alteration in helical parameters and visualisation of thymosin β4 binding on F-actin
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DOI：
10.1006/jmbi.2001.5281
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发表时间：
2002-01-25
影响因子：
5.6
通讯作者：
Mannherz, HG
中科院分区：
文献类型：
--
作者：
Ballweber, E;Hannappel, E;Mannherz, HG
β-胸腺素是细胞内单体 (G-) 肌动蛋白隔离蛋白，与 G-肌动蛋白形成 1:1 复合物。在这里，我们分析了胸腺素β(4) 与F-肌动蛋白的相互作用。 200 muM 的胸腺素 beta(4) 与 F-肌动蛋白化学交联。在鬼笔环肽存在的情况下，化学交联的肌动蛋白：胸腺素β(4)复合物被掺入F-肌动蛋白中。负染色后用传统透射电子显微镜检查时，这些混合细丝具有正常外观。我们纯化了化学交联的肌动蛋白：胸腺素β(4)复合物，该复合物仅在鬼笔环肽和凝溶胶蛋白：2-肌动蛋白复合物同时存在时才聚合。使用扫描透射电子显微镜，发现对照和肌动蛋白：胸腺肽P丝的单位长度质量分别为16.0(+/-0.8) kDa/nm和18.0(+/-0.9) kDa/nm，表明亚基质量增加了5.4 kDa。螺旋参数分析表明，两条右旋长螺距螺旋线的交叉间距从 36.0 nm 增加到 40.5 nm。对照和肌动蛋白：胸腺肽β(4)丝的3D螺旋重建的差异图分析产生了细长的额外质量。定性地，差异质量的总体大小和形状与已发表的胸腺素β(4)原子结构数据一致。推导的胸腺肽β(4) 与肌动蛋白的结合位点与之前确定的一致。然而，差异图的部分可能代表了两种蛋白质在复杂形成时发生的微妙构象变化。 (C) 2002 年学术出版社。
The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 muM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin P, filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation. (C) 2002 Academic Press.