Expression Studies of Myosin X

肌球蛋白X是一种非传统的肌球蛋白，与哺乳动物的丝状突发育有关。我们最近表征了它的稳态和暂态镁ATPase活性。肌球蛋白X含有一个由120个残基组成的预测卷曲区域。然而，高电荷的性质，以及近36个残基中的电荷模式，似乎与盘绕形成不相容。圆二色谱、核磁共振和分析超速离心法表明，含有该区域的合成肽在溶液中形成稳定的单一a-螺旋(SAH结构域)，即使在毫摩尔浓度下也不会二聚形成卷曲。此外，含有马达、三个钙调蛋白结合区和全长预测卷曲的重组肌球蛋白X的电子显微镜显示，在生理蛋白浓度下，它大多是单体。在二聚体中，分子只在其最远端连接，看不到卷曲的尾巴。此外，从钙调蛋白结合域的数量来看，单体和二聚体的颈长都比预期的要长得多。相反，在相同条件下获得的肌球蛋白V HMM的显微照片清楚地显示了卷曲的尾巴，脖子是预测的长度。因此，预测的肌球蛋白X卷曲形成了一种新的细长结构，其中近端是SAH结构域，而远端是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，在预测的肌球蛋白VI、VIIa和myoM的卷曲卷曲结构域中可能存在类似的结构，并可以增加工作卒中的大小。 我们已经设计了一种嵌合肌球蛋白，其中肌球蛋白V的运动区和前两个IQ区域与预测的肌球蛋白V的SAH结构域融合在一起，然后是肌球蛋白V的卷曲线圈杆。我们发现，这种嵌合分子在体外单分子运动分析中是高度进行性的，并且具有更大的步长，这在只有2IQ基序的分子中是可以预测的。旋转阴影电子显微镜显示，添加的SAH结构域延长了颈部的长度。这表明，嵌合体中的SAH结构域可能是杠杆臂的延伸，或者至少是一条灵活的系绳，允许自由头在寻找结合位点的过程中探索更多的肌动蛋白细丝。 我们已经对肌球蛋白X结构进行了光学捕获纳米测量，其中在SAH结构域的末端包括一个亮氨酸拉链来强制分子的二聚化。我们正在测量PowerStroke的长度，分子的硬度，以及分子是否可以连续运动。

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.