EXPRESSION OF STUDIES OF MYOSIN V

平滑肌和非肌肉肌球蛋白II是 由20 kDa调节轻链的磷酸化调节 (RLC)位于颈部。此区域包含单个 肌球蛋白重链的α-螺旋片段和RLC和 必需轻链(ELC)，被称为调节域。 此前的研究已经注意到，像S一号这样的单头物种 和单头肌球蛋白的制备 无论处于何种状态，肌球蛋白都具有很高的镁ATPase活性 RLC磷酸化。然而，这两个物种都有一个 监管领域。以确定是否存在相互作用 肌球蛋白的两个头部的相邻调控结构域是 对于监管至关重要，我们已经制作了一个单头HMM-Like 使用杆状病毒的含有两个调节结构域的分子 非肌肉肌球蛋白IIB片段的表达。Sf9细胞 同时感染了三种病毒。一个编码的和 含有残基1-1263的HMM长度片段。第二 编码残基816-1263在C末端用 国旗表位。此片段绑定RLC和ELC，并 形成盘绕线圈二聚体。第三种病毒表达RLC和RLC 和ELC。标志亲和柱和标志亲和柱的组合 ATP依赖的肌动蛋白结合被用来纯化 异二聚体单头分子。非变性凝胶和 旋转跟踪扫描电子显微镜证实了一块单头碎片 已经做好了准备。其稳定的肌动蛋白激活的镁ATPase 被RLC磷酸化激活3-7倍。自稳态以来 测量结果通常低估了肌球蛋白的程度 规定，我们目前采用的是单周转动态化 测定药物释放速率常数的实验研究 关闭状态。这些研究表明，没有一个单头的 框架蛋白和双头片段一样受到监管，但 单头车架的周转率 完整的调控结构域仍被激活10倍 磷酸化。这表明单头制剂 具有异质动力学，并且同时具有快相和慢相 它的ATPase活性。我们目前使用的是停流 用荧光法测量快相的速度和幅度 这两个阶段。我们实验室以前的工作表明， 非肌肉肌球蛋白IIA和肌球蛋白IIB具有不同的动力学和 能动的特性。在另一项研究中，我们是工程学 杆状病毒肌球蛋白IIA和肌球蛋白IIB S-1样片段 表情。这些碎片应该可以让我们确定 各中间步骤的动力学速率和平衡常数 参与了镁三磷酸腺苷的水解酶。

Smooth muscle and nonmuscle myosin II are regulated by phosphorylation of the 20 kDa regulatory light chain (RLC) located in the neck region. This region contains a single alpha-helical segment of the myosin heavy chain and the RLC and the essential light chain (ELC) and is called the regulatory domain. Previous studies have noted that single-headed species such as S-1 and single-headed myosin prepared by proteolysis of smooth muscle myosin have a high MgATPase activity regardless of the state of RLC phosphorylation. However, both of the species have a single regulatory domain. To determine whether interactions between adjacent regulatory domains of the two heads of myosin are essential for regulation, we have made a single-headed HMM-like molecule containing two regulatory domains using baculoviral expression of nonmuscle myosin IIB fragments. Sf9 cells were simultaneously infected with three viruses. One encoded an HMM-length fragment containing residues 1-1263. The second encoded residues 816-1263 tagged at the C-terminus with the FLAG epitope. This fragment binds both the RLC and ELC and forms a coiled-coil dimer. The third virus expressed both the RLC and ELC. A combination of FLAG affinity column and ATP-dependent binding to actin was used to purify the heterodimeric single-headed molecule. Nondenaturing gels and rotary shadowing EM confirmed that a single-headed fragment had been prepared. Its steady-state actin-activated MgATPase was activated 3-7-fold by RLC phosphorylation. Since steady-state measurements typically underestimate the degree of myosin's regulation, we are currently using single-turnover kinetic experiments to determine the rate constant for product release in the off state. These studies reveal that none of the single-headed framgents are as well regulated as the double-headed fragment, but that the turnover rate of a single-headed frament containing an intact regulatory domain was still activated 10-fold by phosphorylation. This suggests that the single-headed preparation has heterogenous kinetics and has both a fast and a slow phase to its ATPase activity. We are currently using stopped-flow fluorimetry to measure the rate of the fast phase and the amplitudes of both phases. Previous work in our lab has revealed that nonmuscle myosin IIA and myosin IIB have differing kinetic and motile properties. In a separate line of studies, we are engineering myosin IIA and myosin IIB S-1-like fragments for baculoviral expression. These fragments should allow us to determine the kinetic rate and equilibrium constants for various intermediate steps involved in the hydrolysis of MgATP.