Joel Peteranne B, Fagnant Patricia M, Trybus Kathleen M
Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vermont 05405-0068, USA.
Biochemistry. 2004 Sep 14;43(36):11554-9. doi: 10.1021/bi048899a.
We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.
我们已成功在杆状病毒/Sf9细胞系统中高产表达肌动蛋白。野生型（WT）肌动蛋白在激活ATP酶活性以及在体外运动分析中支持运动的能力方面，与组织纯化的肌动蛋白在功能上并无差异。实现这一成果后，我们采用突变策略来表达一种无法聚合的单体肌动蛋白。天然肌动蛋白需要肌动蛋白结合蛋白或化学修饰才能维持在单体状态。在100 mM KCl和2 mM MgCl₂条件下，突变型肌动蛋白在分析超速离心机中以3.2 S的均匀单体形式沉降，而在相同条件下WT肌动蛋白会发生聚合。导致肌动蛋白不可聚合的两个点突变位于4亚结构域（A204E/P243K；“AP-肌动蛋白”），远离肌球蛋白结合位点。AP-肌动蛋白与骨骼肌肌球蛋白亚片段1（S1）结合并形成均匀复合物，这已通过分析超速离心得到证实。交联的AP-肌动蛋白·S1复合物的ATP酶活性高于单独的S1，尽管低于丝状肌动蛋白（F-肌动蛋白）所支持的活性。AP-肌动蛋白是用于肌动蛋白与运动蛋白及其他肌动蛋白结合蛋白复合物结构研究的极佳候选物。
