STRUCTURAL AND FUNCTIONAL ANALYSIS OF DROSOPHILA MYOSIN V

肌球蛋白V是脊椎动物中最具特征的囊泡转运蛋白，但尚不清楚肌球蛋白V家族的所有成员是否具有共同的、进化上保守的作用机制。 我们发现，来自果蝇的肌球蛋白V具有与脊椎动物肌球蛋白Va显著不同的运动机制，并且它是一种非进行性的整体运动。 我们也对肌球蛋白V在果蝇中的作用感兴趣。 为了做到这一点，我们已经定位肌球蛋白V在不同的发展阶段。 初步结果表明，肌球蛋白V在某些类型的细胞定位于核膜，它可能与核纤层蛋白B。 我们正在研制新的抗体，并正在研究肌球蛋白V缺陷突变果蝇核纤层蛋白B的定位。 肌球蛋白V分子的杆片段在这些单分子马达的进行性运动中的作用在很大程度上是未知的。我们比较了进行性（小鼠肌球蛋白Va）和非进行性（果蝇肌球蛋白V）分子马达的机械稳定性。将小鼠肌球蛋白Va和果蝇肌球蛋白V的棒状片段（卷曲螺旋区）分别克隆到原核表达载体pET-16 b中，并在原核表达系统E. coli，BL21（DE3）pLysS）。在变性条件下使用Ni亲和柱纯化重组蛋白。使用圆二色性（CD）监测复性过程中正确卷曲螺旋结构的形成。使用原子力显微镜（AFM）进行单分子力光谱测量。为了促进强和特异性结合，在片段的N-和C-末端应用化学手柄。在机械拉伸过程中，杆片段的延伸作为两个连续的事件发生：柔性环的延伸，然后是卷曲螺旋图案的延伸和展开。我们发现，进行性和非进行性杆碎片显示不同的展开模式。卷曲螺旋结构的展开发生在AFM拉伸周期进行性肌球蛋白Va比非进行性果蝇肌球蛋白V。此外，我们发现，小鼠肌球蛋白Va的卷曲螺旋结构的展开力高于果蝇肌球蛋白V，这表明前卷曲螺旋区域是机械上更稳定。热诱导变性CD测量显示相同的结果。我们的观察结果表明，在肌球蛋白V分子的情况下，货物和电机之间的系绳由一个机械强大的卷曲螺旋区域结合弹性元件，这种连接可能在维持单分子电机的进行性运动中发挥重要作用。 我们还研究了结构的肌球蛋白V尾区的原子力显微镜和旋转阴影电子显微镜。

Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. We showed that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va and it is a non-processive, ensemble motor. We are also interested in what role myosin V plays in Drosophila. To do this, we have been localizing myosin V in various stages of development. Preliminary results suggest that myosin V in some cell types localizes to the nuclear envelope where it may interact with lamin B. We are in the process of raising new antibodies and are studying the localization of lamin B in myosin V deficient mutant Drosophila. The role of the rod fragment of myosin V molecules in the processive motion of these single molecule motors is largely unknown. We compared the mechanical stability of a processive (mouse myosin Va) and nonprocessive (Drosophila myosin V) molecular motors. Rod fragments (coiled-coil regions) of mouse myosin Va and Drosophila myosin V were cloned into expression vector (pET-16b) and expressed in prokaryotic expression system (E. coli, BL21(DE3)pLysS). Recombinant proteins were purified using Ni-affinity column in denaturing conditions. The formation of proper coiled-coil structure during renaturation was monitored using circular dichroism (CD). Single molecule force spectroscopy measurements were performed using atomic force microscopy (AFM). In order to facilitate strong and specific binding, chemical handles were applied at the N- and C-terminal ends of the fragments. During the mechanical stretching, the extension of rod fragment occurs as two consecutive events: extension of flexible loops followed by extension and unfolding of coiled-coil motifs. We find that the processive and nonprocessive rod fragments display different unfolding patterns. The unfolding of coiled-coil structures occurs much later during the AFM stretch cycle for processive myosin Va than for nonprocessive Drosophila myosin V. Additionally, we find that unfolding forces of the coiled-coil structures of mouse myosin Va is higher then those in Drosophila myosin V, suggesting that the former coiled-coil region is mechanically more stable. Heat-induced denaturation CD measurements show the same result. Our observations suggest that the tether between the cargo and motor in case of myosin V molecules consists of a mechanically strong coiled-coil region combined with elastic elements, and this connection might play an important role in sustaining processive motions of single molecule motors. We have also examined the structure of the myosin V tail region by atomic force microscopy and rotary shadowed electron microscopy.