Molecular contacts between nebulin and actin: cross-linking of nebulin modules to the N-terminus of actin.
Molecular contacts between nebulin and actin: cross-linking of nebulin modules to the N-terminus of actin.
复制标题
星云蛋白和肌动蛋白之间的分子接触：星云蛋白模块与肌动蛋白 N 末端的交联。
DOI：
10.1021/bi961236b
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Wang,K
中科院分区：
文献类型：
--
作者：
Shih,CL;Chen,MJ;Linse,K;Wang,K
NeBulin是一种巨大的肌动蛋白结合蛋白，与肌动蛋白共同延伸，被认为在骨骼肌肌节中形成一种复合细丝。为了了解星云蛋白和肌动蛋白之间的分子相互作用，我们应用化学交联技术来定义肌动蛋白和ND8之间的分子联系，ND8是一个两个模块的星云蛋白片段，通过与G-肌动蛋白和F-肌动蛋白结合来促进肌动蛋白聚合和抑制解聚。丹磺酰-ND8与G-肌动蛋白的荧光滴定结果表明，ND8与G-肌动蛋白形成1：1的络合物，解离常数为4.9μM。用零长度交联剂1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide(EDC)处理后，在不损害肌动蛋白聚合能力的情况下，使ND8−G-肌动蛋白复合体共价交联。末端标记Western印迹及序列和质量分析表明，ND8的赖氨酸5与G-肌动蛋白的两个N-末端的酸性残基发生了交联。类似地，我们已经通过末端标记表明，ND8与F-肌动蛋白的交联发生在肌动蛋白原的N-末端。星云蛋白与肌动蛋白N-末端的结合可能对其影响肌动蛋白聚合的能力具有重要意义。此外，星云蛋白模块与亚域1中肌动蛋白N-末端的关联支持了这样的假设，即星云蛋白包裹在肌动蛋白细丝的外缘，其中S1、原肌球蛋白和几个肌动蛋白结合蛋白已知相互作用。
Nebulin, a giant actin binding protein, coextends with actin and is thought to form a composite thin filament in the skeletal muscle sarcomere. To understand the molecular interactions between nebulin and actin, we have applied chemical cross-linking techniques to define molecular contacts between actin and ND8, a two-module nebulin fragment that promotes actin polymerization and inhibits depolymerization by binding to both G- and F-actin. The formation of a 1:1 complex with a dissociation constant of 4.9 μM between ND8 and G-actin was demonstrated by fluorescence titration of dansyl-ND8 with G-actin. Treatment with a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), cross-linked the ND8−G-actin complex covalently without impairing actin's ability to polymerize. End-labeling Western blot and sequence and mass analyses of purified conjugated peptides revealed the cross-linking between lysine 5 of ND8 and the two N-terminal acidic residues of G-actin. Similarly, we have shown by end-labeling that cross-linking of ND8 to F-actin occurred at the N-terminus of actin protomer. The binding of nebulin to the N-terminus of actin is likely to be significant in its ability to affect actin polymerization. Furthermore, the association of nebulin modules with the actin N-terminus in subdomain 1 supports the hypothesis that nebulin wraps around the outer edges of actin filaments where S1, tropomyosin, and several actin binding proteins are known to interact.