EXPRESSION OF STUDIES OF MYOSIN V

肌球蛋白 V 研究的表达

• 批准号: 6109258
• 负责人: JAMES R. SELLERS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6109258
• 关键词: Baculoviridae; Sf9 cell line; actins; active sites; affinity chromatography; animal extract; calmodulin; cow; enzyme activity; molecular cloning; myosins; peptide library; protein engineering; protein purification; protein structure function

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.

平滑肌和非肌肉肌球蛋白II是 受20 kDa调节轻链磷酸化调节 (RLC)位于颈部。该区域包含一个 肌球蛋白重链的α-螺旋片段和RLC， 必需轻链（ELC），称为调节结构域。 先前的研究指出，单头物种，如S-1 和通过平滑肌的蛋白水解制备的单头肌球蛋白 肌球蛋白具有高的MgATPase活性，无论肌球蛋白的状态如何， RLC磷酸化。然而，这两个物种都有一个单一的 监管领域。为了确定是否存在 肌球蛋白两个头部的相邻调节结构域是 对于监管至关重要，我们已经做出了类似于单头HMM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。sf 9细胞 同时感染三种病毒。一个编码的 含有残基1-1263的HMM长度片段。第二 编码的残基816-1263在C-末端用 FLAG表位。该片段结合RLC和ELC， 形成卷曲螺旋二聚体。第三种病毒既表达RLC， 和ELC。FLAG亲和柱和 ATP依赖性结合肌动蛋白用于纯化 异二聚单头分子。非变性凝胶和 旋转阴影EM证实，一个单头碎片 制备了其稳态肌动蛋白激活的MgATPase是 通过RLC磷酸化激活3-7倍。稳态以来 测量通常低估了肌球蛋白的程度 监管，我们目前使用的是单周转动能 实验，以确定产品释放的速率常数， 关闭状态。这些研究表明，没有一个单头的 fragents和双头片段一样受到很好的调节，但是 一个单头框架的周转率， 完整调节结构域仍然被激活10倍， 磷酸化这表明，单头准备 具有非均相动力学，并且具有快速和缓慢相， ATP酶活性。我们目前使用的是停流 荧光法测量快相速率和振幅 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， 能动性在另一项研究中，我们正在设计 杆状病毒的肌球蛋白IIA和肌球蛋白IIB S-1样片段 表情这些碎片应该能让我们确定 各种中间步骤的动力学速率和平衡常数 参与MgATP的水解。