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。 我们已经设计了一个嵌合的肌球蛋白，其中肌球蛋白V的运动域和前两个IQ区域与预测的肌球蛋白V的SAH结构域融合在一起，紧接着是肌球蛋白V的卷曲线圈杆。我们在体外单分子运动分析中发现这种嵌合分子是高度进行性的，并且具有更大的步长，这对于只有2IQ基序的分子来说是可以预测的。这表明嵌合体中的SAH结构域可能是杠杆臂的延伸，或者至少是形成了一条灵活的系绳，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。

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 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.