Expression Studies of Other Unconventional Myosins

其他非常规肌球蛋白的表达研究

• 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
• 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

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。 我们已经表明，该结构域形成一个稳定的单α-螺旋结构域（SAH结构域），并发挥延伸肌球蛋白颈部区域的功能，该区域形成肌球蛋白杠杆臂的一部分。 因此，动力冲程延长。 我们已经进行了光捕获实验，其中亮氨酸拉链被添加在预测的卷曲螺旋区域的末端的肌球蛋白X的强制二聚体。 电子显微镜显示该分子为二聚体。 我们已经测量了它的机械性能，使用光阱纳米测量，发现它有一个约17 nm的功率冲程。 增加钙离子浓度使功率冲程大小增加到23 nm，这与杠杆臂中的三个IQ基序和SAH域一致。 我们认为，增加功率冲程长度是由于在钙的存在下，额外的钙调素结合到第三智商基序。 附着寿命与体外测得的ADP释放速率一致。 在低陷阱刚度，肌球蛋白X显示进行性运动（向前和向后的步骤）发生约35 nm的步骤。 这与电子显微照片一致，显示分子通过两个头连接到肌动蛋白单体，在肌动蛋白丝中相隔36 nm。 与前博士后Takeshi Sakamoto合作，我们发现单分子TIRF测定表明，在没有负载的情况下，分子以36 nm的步长沿沿着肌动蛋白移动。 我们研究了肌球蛋白X在平行束上的运动。 肌球蛋白主要沿着单个肌动蛋白丝行走，但也经常向邻近的肌动蛋白丝侧走 全长肌球蛋白18 A不形成细丝，而是存在于单体和反平行二聚体之间的平衡中。 当肌球蛋白18 A与非肌球蛋白IIA混合时，这两种分子共聚合形成杂聚丝，随着肌球蛋白18 A：肌球蛋白IIA比例的增加，杂聚丝变得更短。在高比率下，没有看到细丝，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研究肌球蛋白5 HMM在肌动蛋白上的进行性运动。 使用这种技术，我们能够成像单个的，未标记的肌球蛋白5 HMM分子沿沿着肌动蛋白移动，精度为几纳米。 该分子以36 nm的步长移动，并以与先前报道的荧光标记肌球蛋白5相同的速度移动。 通过将20 nm的金颗粒连接到氨基末端，我们能够以高达1000 Hz的采样率测量运动，并跟踪未连接的标记肌球蛋白头部的运动。 有趣的是，即使有一个20纳米的金颗粒连接的肌球蛋白以相同的速度移动的未标记的分子。 肌球蛋白3B是一种单体肌球蛋白，我们已经在Sf 9细胞中与钙调蛋白和调节轻链一起沿着表达。 它结合调节轻链和钙调蛋白，如在不存在钙的情况下纯化的。钙调素取代的监管轻链在钙的存在下，这是伴随着肌动蛋白激活的MgATP酶活性的显着增加。