Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
J Mol Biol. 2012 Feb 24;416(3):414-24. doi: 10.1016/j.jmb.2011.12.040. Epub 2011 Dec 28.
Dystrophin is an actin binding protein that is thought to stabilize the cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule [from N-terminal actin binding domain (ABD) 1 through ABD2] bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays than full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction by restricting actin rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.
肌营养不良蛋白是一种肌动蛋白结合蛋白，它被认为在收缩过程中稳定心脏和骨骼肌细胞膜。在这里，我们研究了肌营养不良蛋白的每个结构域对肌动蛋白结合功能的贡献。共沉淀测定和芘肌动蛋白荧光实验证实，跨越肌营养不良蛋白分子三分之二的片段[从 N 端肌动蛋白结合结构域（ABD）1 到 ABD2]与肌动蛋白丝具有高亲和力，并保护丝免受强制解聚，但在两种测定中都不如全长肌营养不良蛋白有效。虽然编码肌营养不良蛋白 C 端三分之一的构建体显示没有特定的肌动蛋白结合活性或与全长肌营养不良蛋白竞争，但我们的数据表明，当存在顺式时，它赋予肌营养不良蛋白 N 端三分之二的肌动蛋白结合的意外调节。时间分辨磷光各向异性实验表明，肌营养不良蛋白 C 端三分之一的存在也通过限制肌动蛋白旋转幅度来影响肌动蛋白相互作用。我们提出，肌营养不良蛋白的 C 端区域通过变构稳定肌营养不良蛋白的最佳肌动蛋白结合构象。
