EXPRESSION OF STUDIES OF MYOSIN V

肌凝蛋白V是一种双头的非常规肌凝蛋白，由于存在六个轻链结合iq重复序列而具有延长的颈部。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，每个片段在其羧基末端标记有flag表位，在Sf9细胞中表达。用含有适当重链、钙调蛋白和必需轻链的三种杆状病毒共同感染细胞。用flag亲和柱层析纯化表达蛋白。肌凝蛋白V片段具有较高的Vmax和较低的稳态肌动蛋白活化的MgATPase。钙对MgATPase活性没有激活作用。与兔骨骼肌肌球蛋白片段相比，KATPase对离子强度不敏感。同样，在体外运动试验中，肌凝蛋白V可以在很宽的离子强度范围内易位肌动蛋白丝，并且可以在非常低的表面密度下易位。ADP明显抑制肌动蛋白激活的MgATPase活性和体外运动。在200mM的ATP中，滑动速度被抑制到25微摩尔ADP时最大滑动速度的50%。瞬态动力学实验表明，在20℃下，ADP以11.5 / s的速率从myosin V亚片段1中解离，在该温度下，肌动蛋白活化的MgATPase活性的Vmax为3.3 / s，表明ADP的解离虽然不是完全限速，但接近限速步骤。对肌动蛋白的高亲和力和缓慢的ADP释放速度有助于确保高占空比，并允许肌动蛋白丝在体外仅被少量肌凝蛋白V分子移动。在细胞内，这意味着只需很少的肌凝蛋白V分子就可以将肌动蛋白丝上的囊泡转运。在与博士合作。英国约克大学的Justin Molloy和Claudia Veigel，我们正在进行单分子力学研究。这些数据表明，肌凝蛋白V每一步能够移动肌动蛋白约20纳米，并且具有很长的附着寿命。此外，该步骤由两个大小不同的子步骤组成。-肌凝蛋白V；体外运动；光学捕获;actin-activated MgATPase;停止流动荧光法

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