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.

项目摘要 这一研究项目的长期目标是了解作用力的分子机制。 通过自然环境中肌球蛋白分子马达的三维可视化。这项研究项目 重点介绍了应用于肌丝结构研究的方法和结果。 大型水虫Lethocerus sp.脊椎动物横纹肌，尤指从 兔，楔形谷盗。我们已经获得了粗丝的近原子分辨率的三维图像。 Lethocerus飞行肌肉，具有4重旋转对称性的螺旋结构。无绕组线圈 肌球蛋白大小的蛋白质以前曾以我们在 肌球蛋白细丝的主干。现在人们对厚角星的三维结构有了更多了解 比任何其他动物的细丝都要多。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。我们现在建议将这些措施延长 方法对难度大得多的脊椎动物骨骼肌粗丝，它不是螺旋状的组装 并且只有三重旋转对称性。狮子座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棒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线圈结构域中。然而， 无脊椎动物的粗丝能在多大程度上告诉人类疾病，取决于它们之间的相似程度 脊椎骨的结构类似于脊椎动物的脊椎动物。在目前的资助期内，我们已获得 史无前例的分辨率和细节的放松状态的粗丝从Lethocerus飞行肌肉。这 肌球蛋白杆突变影响肌肉的机制研究进展提供了机会 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， 隔离肌球蛋白头部与细丝的相互作用。在光滑的和非肌肉的细丝中 肌球蛋白，头部折叠导致细丝不稳定，形成可溶的构象，称为10S， 不能聚合的。这种现象被认为是由于杆结构的变化造成的 这是由头部折叠引起的。简而言之，肌球蛋白杆和肌球蛋白头的结构是耦合的 在某种程度上。最近的肌肉研究表明，无论是通过内部施加的张力，还是通过 肌球蛋白头部或外部受到拉伸，可影响粗大的细丝结构。因此，粗大的长丝 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影响肌球蛋白头的结构，反之亦然， 肌球蛋白头部会影响肌球蛋白尾部的结构。这一假设可以通过以下方式使用CryoEM进行验证 将自然形成的超松弛细丝与肌球蛋白头部紊乱的细丝进行比较。