Isolation, Characterization and Reconstruction of Vertebrate Striated Muscle Myosin Filaments

脊椎动物横纹肌肌球蛋白丝的分离、表征和重建

• 批准号: 10043292
• 负责人: Jose Renato Pinto
• 金额: $ 16.42万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-24 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043292
• 关键词: Affect; Amino Acid Sequence; Animal Model; Animals; Calpain; Cardiac; Charge; Clip; Coiled-Coil Domain; Coupled; Cryoelectron Microscopy; Data Collection; Disease; Drosophila genus; Drosophila melanogaster; Electron Microscope; Environment; Evolution; Fiber; Filament; Funding; Genetic Models; Goals; Head; Human; Image; Insecta; Invertebrates; Length; Location; Measures; Methods; Modeling; Molecular; Molecular Conformation; Molecular Motors; Muscle; Muscle function; Mutation; Myofibrils; Myopathy; Myosin ATPase; Myosin Rod; Myosin Type II; Nemaline Myopathies; Nematoda; Oryctolagus cuniculus; Polymers; Positioning Attribute; Probability; Procedures; Production; Proteins; Publishing; Research; Research Personnel; Research Project Grants; Resolution; Robotics; Rod; Roentgen Rays; Rotation; Science; Sequence Homology; Skeletal Muscle; Skeletal Muscle Myosins; Sodium Chloride; Specimen; Stretching; Striated Muscles; Structure; Tail; Technology; Testing; Thick Filament; Thin Filament; Three-Dimensional Image; Transducers; Ursidae Family; Vertebral column; Vertebrates; Writing; beta-Myosin; detector; human disease; impression; mutant; non-muscle myosin; protein degradation; reconstruction; three dimensional structure; three-dimensional visualization

Project Summary The long term goal of this research project is to understand the molecular mechanism of force production through 3-D visualization of myosin molecular motors in their natural environment. This research project focuses on extending methods used and results obtained in structural studies of muscle filaments isolated from the large waterbug Lethocerus sp. to vertebrate striated muscle, specifically skeletal muscles obtained from rabbits, Oryctolagus cuniculus. We have obtained a near atomic resolution 3-D image of thick filaments from Lethocerus flight muscle, which have a helical structure with 4-fold rotational symmetry. No coiled-coil protein of the size of myosin had been imaged previously at the resolution we have achieved (4.2Å) in the backbone of the myosin filament. There is now more known about the 3-D structure of Lethocerus thick filaments than those from any other animal. Recent advancements in detector technology, robotic electron microscopes and high throughput data collection, have made this possible. We now propose to extend these methods to the much more difficult vertebrate skeletal muscle thick filament, which is not a helical assembly and has only 3-fold rotational symmetry. The high-resolution structure of Lethocerus thick filaments suggests that studies of invertebrate thick filaments can inform familial muscle diseases caused by myosin rod mutations. About 40% of disease-causing myosin mutations occur in the myosin coiled-coil domain. However, how well invertebrate thick filaments can inform human disease depends on how similar their filament backbones are structured like those of vertebrates. In the current funding period, we have obtained unprecedented resolution and detail of the relaxed state of thick filaments from Lethocerus flight muscle. This advance provides opportunity to investigate the mechanism whereby myosin rod mutations can affect muscle function. The head folding of myosin II produces a head conformation called the interacting heads motif that sequesters the myosin heads from interaction with the thin filament. In filaments of smooth and non-muscle myosin, the head folding leads to filament instability and formation of a soluble conformation, called 10S, incapable of polymerizing. This phenomenon has been hypothesized to be due to changes in the rod structure brought on by the head folding. Put simply, the structure of the myosin rod and the myosin heads are coupled in some way. Recent muscle research has pointed to the possibility that tension applied either internally by myosin heads or externally by a stretch, can affect the structure of the thick filament. Thus, the thick filament may function as a tension transducer, but the molecular mechanism by which this occurs is unknown. We hypothesize that tension applied to the thick filament affects the structure of the myosin heads and vice versa, that the myosin heads affect the structure of the myosin tails. This hypothesis can be tested using cryoEM by comparing a naturally formed super-relaxed filament to one with the myosin heads disordered.

项目概要 该研究项目的长期目标是了解力产生的分子机制 通过自然环境中肌球蛋白分子马达的 3D 可视化。本研究项目 重点关注扩展从分离的肌丝的结构研究中使用的方法和获得的结果 大型水蝽 Lethocerus sp.脊椎动物的横纹肌，特别是从获得的骨骼肌 兔子，Oryctolagus cuniculus。我们获得了粗丝的近原子分辨率 3D 图像 Lethocerus飞行肌，具有四重旋转对称的螺旋结构。无线圈 肌球蛋白大小的蛋白质之前已以我们已达到的分辨率 (4.2Å) 进行成像 肌球蛋白丝的主干。现在对 Lethocerus 厚层的 3D 结构有了更多的了解 比任何其他动物的细丝都长。探测器技术、机器人电子的最新进展 显微镜和高通量数据收集使这成为可能。我们现在建议延长这些 更困难的脊椎动物骨骼肌粗丝的方法，它不是螺旋组件 并且只有三重旋转对称性。 Lethocerus粗丝的高分辨率结构表明 对无脊椎动物粗丝的研究可以为肌球蛋白杆引起的家族性肌肉疾病提供信息 突变。大约 40% 的致病肌球蛋白突变发生在肌球蛋白卷曲螺旋结构域中。然而， 无脊椎动物的粗丝能否告知人类疾病取决于它们的丝的相似程度 脊柱的结构与脊椎动物相似。在本轮融资期间，我们已获得 来自Lethocerus飞行肌的粗细丝的松弛状态具有前所未有的分辨率和细节。这 进展提供了研究肌球蛋白杆突变影响肌肉的机制的机会 功能。肌球蛋白 II 的头部折叠产生一种称为相互作用头部基序的头部构象 隔离肌球蛋白头与细丝的相互作用。在光滑和非肌肉的细丝中 肌球蛋白，头部折叠导致丝不稳定并形成可溶性构象，称为 10S， 不能聚合。这种现象被假设是由于杆结构的变化造成的 由头部折叠引起。简单来说，肌球蛋白杆和肌球蛋白头的结构是耦合的 以某种方式。最近的肌肉研究指出，张力可能是通过内部施加的 肌球蛋白头部或外部受到拉伸，可影响粗丝的结构。因此，粗丝 可能起到张力传感器的作用，但发生这种情况的分子机制尚不清楚。我们 假设施加在粗丝上的张力会影响肌球蛋白头的结构，反之亦然， 肌球蛋白头部影响肌球蛋白尾部的结构。该假设可以通过冷冻电镜进行检验 将自然形成的超松弛肌丝与肌球蛋白头紊乱的肌丝进行比较。