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首页> 外文期刊>Biochemistry >Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein.
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Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein.

机译：盘基网柄菌34,000道尔顿肌动蛋白捆绑蛋白中的三个不同的F-肌动蛋白结合位点。

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The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.
机译：Dictyostelium 34 kDa蛋白是一种肌动蛋白捆绑蛋白，由295个氨基酸组成。但是，结合肌动蛋白丝的分子的区域是未知的。对125I-34 kDa蛋白和F-肌动蛋白的共沉淀研究表明，对于4.9微米和0.6微米的长丝，该34 kDa蛋白以正协同性和Hill系数分别为1.9和3.0结合F-肌动蛋白。对于短丝而言，比长丝更有效地捆扎的希尔系数更大，这表明结合位点之一用于丝间接触或有助于丝束在丝束内定向。使用合成肽，蛋白质截断和新颖的表位库筛选方法确定了三个不同的肌动蛋白结合位点。结合肌动蛋白的能力是通过在变性和非变性条件下，共沉淀，粘度测定和pyr标记的肌动蛋白拆卸下125 I-F-肌动蛋白覆盖层评估的。三个肌动蛋白结合结构域被鉴定为氨基酸1-123、193-254和279-295。 62个氨基酸的结构域（193-254）可以与F-肌动蛋白共沉淀。通过分解pyr标记的肌动蛋白获得的估计Kapp对氨基酸1-123和279-295分别为0.11 microM和2.7 microM。这些结果确定了34 kDa蛋白的三个不同区域，这些区域可能有助于F-肌动蛋白束的正性协作形成。

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