Expression Studies of Other Unconventional Myosins

其他非常规肌球蛋白的表达研究

基本信息

批准号：
8939785
负责人：
James Sellers
金额：
$ 78.18万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/8939785
关键词：
Actins Behavior Binding Biochemical Biological Assay Calcium Calmodulin Cells Charge Collaborations Development Electron Microscopy Electrons Equilibrium Filament Goals Gold Head Image Image Analysis In Vitro Kinetics Label Length Leucine Zippers Light Mammals Measures Mechanics Microfilaments Microscopic Molecular Movement Myosin ATPase Nature Neck Nonmuscle Myosin Type IIA Pattern Postdoctoral Fellow Power stroke Property Proteins Regulation Reporting Sampling Side Solutions Speed Structure Techniques Universities Walking arm base biophysical techniques dimer interest light microscopy member monomer nanometer non-muscle myosin optical traps particle protein structure research study single molecule

Actins Behavior Binding Biochemical Biological Assay Calcium Calmodulin Cells Charge Collaborations Development Electron Microscopy Electrons Equilibrium Filament Goals Gold Head Image Image Analysis In Vitro Kinetics Label Length Leucine Zippers Light Mammals Measures Mechanics Microfilaments Microscopic Molecular Movement Myosin ATPase Nature Neck Nonmuscle Myosin Type IIA Pattern Postdoctoral Fellow Power stroke Property Proteins Regulation Reporting Sampling Side Solutions Speed Structure Techniques Universities Walking arm base biophysical techniques dimer interest light microscopy member monomer nanometer non-muscle myosin optical traps particle protein structure research study single molecule

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项目摘要

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. We have shown that this domain forms a stable single alpha-helical domain (SAH domain) and functions to extend the neck region of the myosin which forms part of the lever arm of myosin. Thus, the powerstroke is lengthened. We have carried out optical trapping experiments with a forced dimer of myosin X where a leucine zipper was added at the end of the predicted coiled-coil region. This molecule is shown to be dimeric by electron microscopy. We have measured its mechanical properties using optical trapping nanometry and find that it has a power stroke of about 17 nm. Increasing the calcium concentration increased the power stroke size to 23 nm consistent with three IQ motifs and a SAH domain in the lever arm. We believe the increase power stroke length is due to binding of an additional calmodulin to the third IQ motif in the presence of calcium. The attachment lifetimes are consistent with the ADP release rate measured in vitro. At low trap stiffness, the myosin X shows processive movement (forward and backward steps) occurring with steps of about 35 nm. This is consistent with electron micrographs showing the molecule attached by two head to actin monomer that are separated by 36 nm in the actin filament. In collaboration with a former postdoc, Takeshi Sakamoto, we show that single molecule TIRF assays show that the molecule moves processively along actin in the absence of load with 36 nm steps. We examined the movement of myosin X on parallel bundles. The myosin walks predominantly along a single actin filament, but takes frequent side steps onto adjacent filaments Full length myosin 18A does not form filaments, but rather exists in an equilibrium between a monomer and an antiparallel dimer. When myosin 18A is mixed with nonmuscle myosin IIA, the two molecules co-polymerize to form heteropolymeric filaments which become shorter as the ratio of myosin 18A:myosin IIA increases. At high ratios, no filaments are seen, but rather dimeric molecules in solution. In collaboration with Philipp Kukura of Oxford University, we have used a light microscopy based interferometric scattering technique to examine the processive movement of myosin 5 HMM on actin. Using this technique we were able to image single, unlabeled molecules of myosin 5 HMM move along actin with a precision of a few nanometers. The molecule took 36 nm steps and moved at the same speed as previously reported for fluorescently-labeled myosin 5. By attaching a 20 nm gold particle to the amino-terminus we are able to measure the movement at sampling rates up to 1000 Hz and follow the movement of the unattached labeled myosin head. Interesting, even with a 20 nm gold particle attached the myosin moves at the same velocity as the unlabeled molecule. Myosin 3B is a monomeric myosin which we have expressed in Sf9 cells along with calmodulin and regulatory light chain. It binds regulatory light chain and calmodulin as purified in the absence of calcium. Calmodulin displaces the regulatory light chain in the presence of calcium and this is accompanied by a significant increase in the actin-activated MgATPase activity.

肌球蛋白X是一种非常规的肌球蛋白，与哺乳动物的丝虫发育有关。我们最近表征了其稳态和瞬态态MGATPase活性。肌球蛋白X包含一个预测的盘绕螺旋的区域120个残基。然而，近端36个分配中高度带电的性质和电荷的模式似乎与盘绕螺旋形成不相容。我们已经表明，该结构域形成稳定的单α-螺旋结构域（SAH结构域）和功能，以扩展肌球蛋白的颈部区域，肌球蛋白的颈部区域构成肌球蛋白杆的一部分。因此，力量延长了。我们已经使用肌球蛋白X的强制二聚体进行了光学诱捕实验，在该二聚体X的末端添加了亮氨酸拉链。通过电子显微镜证明该分子是二聚体。我们已经使用光学诱捕纳米测量了其机械性能，并发现其具有约17 nm的功率行程。钙浓度的增加将功率冲程大小提高到23 nm，与三个智商基序和杠杆臂中的一个SAH结构域一致。我们认为，动力冲程长度的增加是由于在钙存在下额外的钙调蛋白与第三个智商基序结合。附着寿命与体外测得的ADP释放率一致。在低陷阱刚度时，肌球蛋白X显示了大约35 nm的步骤发生的过程（向前和向后步骤）。这与电子显微照片是一致的，显示了两个头部与肌动蛋白单体相连的分子，这些分子在肌动蛋白丝中被36 nm分离。通过与以前的博士后Takeshi Sakamoto合作，我们表明单分子TIRF测定法表明，该分子在没有36 nm步骤的情况下沿肌动蛋白进行过程移动。我们检查了肌球蛋白X在平行束上的运动。肌球蛋白主要沿着单个肌动蛋白细丝行走，但频繁的侧面步骤在相邻的丝上，全长肌球蛋白18a不会形成丝，而是形成丝，而是在单体和对二二聚体之间的平衡中存在。当肌球蛋白18a与非肌肉肌球蛋白IIA混合时，这两个分子共聚合形成异质符号丝，随着肌球蛋白18a的比率较短，肌球蛋白IIA的比例会增加。在高比率下，没有看到丝，而是溶液中的二聚体分子。与牛津大学的Philipp Kukura合作，我们使用了基于光学显微镜的干涉散射技术来检查肌球蛋白5 HMM在肌动蛋白上的过程运动。使用这种技术，我们能够以几种纳米的精确度对肌球蛋白5 hmm的单个未标记的分子进行成像。该分子采取了36 nm步骤，并以与先前报道的荧光标记的肌球蛋白5相同的速度移动。通过将20 nm的金粒子连接到氨基末端，我们能够以高达1000 Hz的采样速率测量运动，并遵循未附加标记的肌球蛋白头的运动。有趣的是，即使有20 nm的金颗粒，肌球蛋白也以与未标记的分子相同的速度移动。肌球蛋白3b是一种单体肌球蛋白，我们在SF9细胞中与钙调蛋白和调节轻链一起表达。在没有钙的情况下，它结合了调节轻链和钙调蛋白。钙调蛋白在钙的存在下置换了调节光链，这伴随着肌动蛋白激活的MGATPase活性的显着增加。