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

基本信息

批准号：
10268975
负责人：
Jose Renato Pinto
金额：
$ 19.21万
依托单位：
FLORIDA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-24 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10268975
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

项目摘要

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。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. 脊椎动物横纹肌，特别是从其获得的骨骼肌我们获得了来自兔子的粗丝的近原子分辨率 3D 图像。 Lethocerus 飞行肌，具有 4 重旋转对称的螺旋结构，无卷曲螺旋。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) 进行成像现在对 Lethocerus 粗体的 3-D 结构有了更多的了解。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，我们现在建议扩展这些。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仅具有 3 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。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骨干的结构与脊椎动物相似，在当前的资助期内，我们已经获得了这些骨干的结构。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。提供了研究肌球蛋白杆突变影响肌肉的机制进展的机会肌球蛋白 II 的头部折叠产生称为相互作用头部基序的头部构象。隔离肌球蛋白头与平滑肌丝和非肌肉肌丝的相互作用。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，这种现象被认为是由于棒结构的变化造成的。简单地说，肌球蛋白杆和肌球蛋白头的结构是耦合的。在某种程度上，最近的肌肉研究指出张力可能是通过内部施加的。肌球蛋白头或外部受到拉伸，可影响粗丝的结构，从而形成粗丝。可能起到张力传感器的作用，但其发生的分子机制尚不清楚。保持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使用冷冻电镜进行检验。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。