Isolation, Characterization and Reconstruction of Vertebrate Striated Muscle Myosin Filaments

项目摘要 本研究项目的长期目标是了解力产生的分子机制 通过对肌球蛋白分子马达在自然环境中的三维可视化。本研究项目 着重于扩展所用的方法和结果中获得的肌丝分离的结构研究， 将大型水蝽Lethocerus sp.施用到脊椎动物横纹肌，特别是从 兔子Oryctolagus cuniculus我们已经获得了一个近原子分辨率的3-D图像的粗丝， Lethocerus飞行肌，具有4重旋转对称的螺旋结构。无螺旋线圈 肌球蛋白大小的蛋白质先前已经以我们在2008年获得的分辨率（4.2 μ m）成像。 肌球蛋白丝的骨架。现在有更多的了解三维结构的Lethocerus厚 比其他任何动物都要多。探测器技术的最新进展，机器人电子 显微镜和高通量数据收集使这成为可能。我们现建议将这些 方法要困难得多的脊椎动物骨骼肌粗丝，这是不是一个螺旋组装 并且只有三重旋转对称性。白屈菜属粗纤维的高分辨率结构表明 对无脊椎动物粗肌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。然而，在这方面， 无脊椎动物粗纤维能够告诉人类疾病的程度取决于它们的纤维的相似程度 脊椎的结构与脊椎动物相似。在目前的融资期内，我们获得了 前所未有的分辨率和细节的松弛状态的粗丝从Lethocerus飞行肌肉。这 这一进展为研究肌球蛋白杆突变影响肌肉的机制提供了机会 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， 隔离肌球蛋白头与细丝的相互作用。在平滑肌和非肌纤维中 肌球蛋白，头部折叠导致细丝不稳定和形成可溶性构象，称为10 S， 不能聚合。这种现象被假设是由于杆结构的变化 是由头部折叠引起的简单地说，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已经指出，紧张的可能性，无论是内部施加的， 肌球蛋白头部或外部受到拉伸，可影响粗肌丝的结构。因此， 可以作为张力传感器，但发生这种情况的分子机制是未知的。我们 假设施加于粗丝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。该假设可以使用cryoEM进行测试， 将自然形成的超松弛肌丝与肌球蛋白头部紊乱的肌丝进行比较。

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.