Expression Studies of Myosin X

肌凝蛋白X是一种非常规的肌凝蛋白，与哺乳动物丝状体发育有关。我们最近对其稳态和瞬态MgATPase活性进行了表征。Myosin X包含一个预测的120个残基长的螺旋状区域。然而，高电荷性质和近36个残基的电荷模式似乎与盘绕线圈形成不相容。圆二色性、核磁共振和分析性超离心表明，含有该区域的合成肽在溶液中形成稳定的单a-螺旋（SAH结构域），即使在毫摩尔浓度下也不会二聚化形成螺旋状。此外，含有马达、三个钙调蛋白结合结构域和全长预测线圈的重组肌球蛋白X的电镜显示，在生理蛋白浓度下，重组肌球蛋白X大部分是单体的。在二聚体中，分子仅在其极端远端连接，没有可见的盘绕尾。此外，单体和二聚体的颈长度都比钙调素结合域的数量预期的要长得多。相比之下，在相同条件下获得的myosin V HMM显微照片清楚地显示出卷曲的尾巴，颈部为预测长度。因此，预测的肌球蛋白X的卷曲形成了一种新的细长结构，其中近端区域是SAH结构域，远端区域是SAH结构域（或具有未知的扩展结构），仅在其末端二聚化。序列比较表明，在肌凝蛋白VI、VIIa和myoM的预测线圈结构域中可能存在类似的结构，并可能起增加工作行程大小的作用。

Myosin X is an unconventional myosin that has been implicated in filopodial development in mammals. We have recently characterized its steady-state and transient state MgATPase activity. Myosin X contains a region of predicted coiled-coil 120 residues long. However, the highly charged nature, and pattern of charges in the proximal 36-residues, appears incompatible with coiled-coil formation. Circular dichroism, NMR and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single a-helix (SAH domain) in solution and does not dimerize to form coiled-coil, even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin X containing the motor, the three calmodulin binding domains and the full-length predicted coiled-coil showed that it was mostly monomeric at physiological protein concentration. In dimers, the molecules were only joined at their extreme distal ends and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin V HMM obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus, the predicted coiled-coil of myosin X forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins VI, VIIa, and myoM, and could function to increase the size of the working stroke. We have engineered a chimeric myosin in which the motor domain and first two IQ regions of myosin V are fused with the predicted SAH domain of myoM followed by the coiled-coil rod of myosin V. We have found that this chimeric molecule is highly processive in single molecule in vitro motility assays and has a step-size larger that would be predicted for a molecule with only 2IQ motifs. Rotary shadowing electron microscopy reveals that the length of the neck is extended by the added SAH domain. This suggests that the SAH domain engineered into the chimera is possibly acting as an extension of the lever arm or at least makes a flexible tether that allows the free head to explore more of the actin filament in searching for a binding site. We have carried out optical trapping nanometry of a myosin X construct in which a leucine zipper was included at the end of the SAH domain to force dimerization of the molecule. We are measuring the length of the powerstroke, the stiffness of the molecule and whether this molecule can move processively.