Expression Studies of Myosin X

肌球蛋白 X 的表达研究

• 批准号: 7594411
• 负责人: JAMES R. SELLERS
• 金额: $ 22.56万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594411
• 关键词: Altretamine; Binding; Binding Sites; Biological Assay; Calmodulin; Charge; Chimera organism; Circular Dichroism; Coiled-Coil Domain; Condition; Development; Distal; Electron Microscopy; Engineering; Head; Helix (Snails); In Vitro; Length; Mammals; Microfilaments; Motor; Myosin ATPase; Myosin Type II; Myosin Type V; Nature; Neck; Numbers; Pattern; Peptides; Physiological; Proteins; Recombinants; Solutions; Structure; Subarachnoid Hemorrhage; Tail; Testing; Upper arm; Working stroke; alpha helix; analytical ultracentrifugation; cell motility; dimer; ear helix; monomer; myosin VI; novel; retinal rods; single molecule; size

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.

肌球蛋白X是一种非传统的肌球蛋白，与哺乳动物的丝状伪足发育有关。 最近，我们的特点是其稳态和瞬态MgATPase活性。 肌球蛋白X包含一个预测的卷曲螺旋120个残基长的区域。 然而，高度带电的性质，并在近端36-残基的电荷模式，似乎与卷曲螺旋的形成不兼容。 圆二色性、NMR和分析超离心表明，含有该区域的合成肽在溶液中形成稳定的单个α-螺旋（SAH结构域），并且即使在毫摩尔浓度下也不会二聚形成卷曲螺旋。 此外，电子显微镜的重组肌球蛋白X含有电机，三个钙调蛋白结合域和全长预测卷曲螺旋显示，它主要是单体在生理蛋白浓度。 在二聚体中，分子仅在其最远端连接，并且看不到卷曲螺旋尾。 此外，无论是单体和二聚体的颈部长度远长于预期的钙调蛋白结合结构域的数量。 相比之下，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螺旋尾，并且颈部是预测的长度。 因此，预测的卷曲螺旋肌球蛋白X形成一种新的细长结构，其中近端区域是SAH结构域，远端区域是SAH结构域（或具有未知的延伸结构），仅在其末端二聚化。 序列比较表明，类似的结构可能存在于预测的卷曲螺旋结构域的肌球蛋白VI，VIIa，和myoM，并可能发挥作用，以增加工作行程的大小。 我们已经设计了一种嵌合肌球蛋白，其中肌球蛋白V的马达结构域和前两个IQ区域与myoM的预测SAH结构域融合，随后是肌球蛋白V的卷曲螺旋杆。我们发现这种嵌合分子在单分子体外运动试验中具有高度的进行性，并且具有一个步骤-这表明，被工程化到嵌合体中的SAH结构域可能充当杠杆臂的延伸，或者至少形成了一个柔性的允许自由头探索更多肌动蛋白丝以寻找结合位点的系链。