Cross-linking constraints on F-actin structure
Cross-linking constraints on F-actin structure
复制标题
DOI：
10.1006/jmbi.2000.3727
复制
发表时间：
2000-06-02
影响因子：
5.6
通讯作者：
Reisler, E
中科院分区：
文献类型：
--
作者：
Kim, E;Wriggers, W;Reisler, E
DNA酶I结合环（残基38 - 52）、疏水塞（残基262 - 274）以及C末端区域是单体（G -）肌动蛋白的结构元件，据推测它们在F - 肌动蛋白中形成单体间的界面。为了检测这些元件的邻近关系和相互作用，并对F - 肌动蛋白结构模型提供限制条件，在酵母肌动蛋白的残基41或265处引入了半胱氨酸残基。这些突变使得F - 肌动蛋白在C41和C265、C265和C374以及C41和C265之间能够使用二溴二甲基乙内酰脲并通过二硫键形成进行特异性交联。交联产物通过SDS - PAGE和电子显微镜进行观察。对交联的F - 肌动蛋白进行的模型计算表明，在二硫键交联的片段中，肌动蛋白残基需要相当大的柔性或位移，才能使这些细丝符合F - 肌动蛋白的模型结构。计算得到的交联结构与霍姆斯的F - 肌动蛋白模型比与改进的洛伦兹模型更吻合。基于此类计算预测，对二硫键交联的C41 - C374 F - 肌动蛋白的电子显微照片进行图像重建应该能对这两种相似的F - 肌动蛋白结构模型进行决定性的检验。（C）2000学术出版社
The DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274), and the C terminus region are among the structural elements of monomeric (G-) actin proposed to form the intermonomer interface in F-actin. To test the proximity and interactions of these elements and to provide constraints on models of F-actin structure, cysteine residues were introduced into yeast actin either at residue 41 or 265. These mutations allowed for specific cross-linking of F-actin between C41 and C265, C265 and C374, and C41 and C265 using dibromobimane and disulfide bond formation. The cross-linked products were visualized on SDS-PAGE and by electron microscopy. Model calculations carried out for the cross-linked F-actins revealed that considerable flexibility or displacement of actin residues is required in the disulfide cross-linked segments to fit these filaments into model F-actin structures. The calculated, cross-linked structures showed a better fit to the Holmes rather than the refined Lorenz model of F-actin. It is predicted on the basis of such calculations that image reconstruction of electron micrographs of disulfide cross-linked C41-C374 F-actin should provide a conclusive test of these two similar models of F-actin structure.(C) 2000 Academic Press.