EXPRESSION OF STUDIES OF MYOSIN V

肌球蛋白 V 研究的表达

• 批准号: 6290443
• 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/6290443
• 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

Myosin V is a two-headed unconventional myosin that has an extended neck due to the presence of six light chain binding IQ-repeats. Double- headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V, each tagged with the FLAG-epitope on their carboxyl-terminal end, were expressed in Sf9 cells. The cells were coinfected with three baculoviruses containing the appropriate heavy chain, calmodulin and essential light chain. The expressed proteins were purified using FLAG-affinity column chromatography. The myosin V fragments had a high Vmax and a low KATPase of the steady state actin- activated MgATPase. There was no activation of the MgATPase activity by calcium. The KATPase for the fragments were remarkably insensitive to ionic strength compared to fragments of rabbit skeletal muscle myosin. Similarly, myosin V translocated actin filaments over a wide range of ionic strengths in an in vitro motility assay and could do so at very low surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. In 200mM ATP, the sliding velocity was inhibited to 50% of its maximum at 25 micormolar ADP. Transient kinetic experiments revealed that ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 per sec at 20 C. The Vmax of the actin activated MgATPase activity at this temperature was 3.3 per sec, indicating that, while not totally rate limiting, ADP dissociation was close to the rate limiting step. The high affinity for actin and the slow rate of ADP release helps to assure a high duty cycle ratio and allows actin filaments to be moved by only a few myosin V molecules in vitro. Inside the cell, this means that few myosin V molecules would be required to translocate vesicles on actin filaments. In collaboration with Drs. Justin Molloy and Claudia Veigel of University of York, UK, we are performing single molecule mechanical studies. These data indicate that myosin V is able to move actin about 20 nm per step and that it has a very long attached lifetime. In addition, the step is composed of two substeps of dissimilar magnitude. - myosin V; in vitro motility; optical trapping;actin-activated MgATPase; stopped- flow fluorimetry

肌球蛋白V是一种双头非常规肌球蛋白，由于存在六个轻链结合IQ重复序列，因此具有延长的颈部。小鼠肌球蛋白V的双头（重肌球蛋白样）和单头（亚片段1样）片段在Sf 9细胞中表达，每个片段在其羧基末端用FLAG表位标记。用含有适当重链、钙调蛋白和必需轻链的三种杆状病毒共感染细胞。用FLAG-亲和柱层析纯化表达蛋白。肌球蛋白V片段具有稳态肌动蛋白激活的MgATP酶的高Vmax和低KATP酶。钙对MgATPase活性无激活作用。与兔骨骼肌肌球蛋白片段相比，片段的KATP酶对离子强度显着不敏感。同样，肌球蛋白V易位肌动蛋白丝在很宽的范围内的离子强度在体外运动试验，可以这样做，在非常低的表面密度。ADP显著抑制肌动蛋白激活的MgATPase活性和离体运动。在200 mM ATP中，当ADP浓度为25 μ mol时，滑动速度被抑制到最大值的50%。瞬时动力学实验表明，ADP在20 ℃下以约11.5/秒的速率从肌球蛋白V亚片段1上解离。在此温度下，肌动蛋白激活的MgATPase活性的Vmax为每秒3.3，表明虽然不完全限速，但ADP解离接近限速步骤。对肌动蛋白的高亲和力和ADP释放的缓慢速率有助于确保高占空比，并允许肌动蛋白丝在体外仅被少数肌球蛋白V分子移动。在细胞内，这意味着几乎不需要肌球蛋白V分子就可以将囊泡转移到肌动蛋白丝上。我们与英国约克大学的Justin Molloy和Claudia Veigel博士合作，进行单分子力学研究。这些数据表明，肌球蛋白V是能够移动肌动蛋白约20纳米每一步，它有一个很长的附着寿命。此外，该步骤由大小不同的两个子步骤组成。- 肌球蛋白V;离体运动;光捕获;肌动蛋白激活的镁ATP酶;停流荧光法