EXPRESSION OF STUDIES OF MYOSIN V

肌球蛋白V是一种双头非传统肌球蛋白，由于存在六个轻链结合IQ重复序列，其颈部延长。在Sf9细胞中表达了小鼠肌球蛋白V的双头(重链肌球蛋白样)和单头(亚段1样)片段，每个片段的羧基末端都标记了标志表位。细胞与含有适当重链、钙调蛋白和必需轻链的三种杆状病毒共感染。用FLAG亲和柱层析对表达蛋白进行纯化。肌球蛋白V片段具有高Vmax和低KATPase的稳态肌动蛋白激活的镁ATPase。钙不能激活镁ATP酶的活性。与兔骨骼肌肌球蛋白片段相比，这些片段的KATPase对离子强度明显不敏感。类似地，肌球蛋白V在体外运动性分析中可以在广泛的离子强度范围内转移肌动蛋白细丝，并且可以在非常低的表面密度下做到这一点。ADP明显抑制肌动蛋白激活的镁ATPase活性和体外运动。在200 mM的三磷酸腺苷中，当ADP浓度为25微摩尔时，滑动速度被抑制到最大值的50%。瞬时动力学实验表明，在20℃下，ADP与肌球蛋白V亚段1的解离速率约为11.5/s。在此温度下，肌动蛋白激活的镁ATPase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。肌动蛋白的高亲和力和ADP的缓慢释放速度有助于确保高占空比，并允许肌动蛋白细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分子来转移肌动蛋白细丝上的小泡。与英国约克大学的贾斯汀·莫洛伊博士和克劳迪娅·维格尔博士合作，我们正在进行单分子力学研究。这些数据表明，肌球蛋白V能够每步移动约20 nm的肌动蛋白，并且它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。-肌球蛋白V；体外运动；光学捕获；肌动蛋白激活的镁ATPase；停流荧光法

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