Myofibrillar Structure of Striated Muscle

该项目的长期目标是了解肌肉如何将化学能转化为机械力，运动和工作。所有的肌肉都通过运动蛋白肌球蛋白，周期性地水解ATP和拉动肌动蛋白丝来收缩。肌球蛋白和肌动蛋白各自的原子结构是已知的，但肌球蛋白在拉动肌动蛋白时所经历的结构变化仍然知之甚少。我们的工作旨在直接可视化，在准原子水平上，肌球蛋白的结构变化的全方位 在工作中经历。最有趣的状态，即当肌球蛋白产生力时，是固有的动态的，但可以在肌节内进行冷冻捕获和研究，肌节是特别进化的粗丝和细丝的晶格，其将肌球蛋白和肌动蛋白保持在紧密接近和高的位置。 密度的我们的方法在全球范围内仍然是独一无二的，并集成了肌肉生理学，X射线衍射（XRD），快速（~1 ms）冷冻保存，电子显微镜，使用电子断层扫描（EM/ET）的3D重建，类平均和准原子模型构建使用Lethocerus的飞行肌肉，其近结晶的细丝晶格使其成为此类结构研究的最佳标本。由于单个肌球蛋白彼此不同步，稳态收缩自动包含反映生化平衡动力学的所有可能的肌球蛋白状态的完整集合，对生化或机械扰动和研究开放。肌肉生理学和XRD报告动态集合平均值的功能和结构状态，并通过快速冷冻捕获和EM/ET进行补充，使我们能够直接以3D方式可视化整个集合。多变量数据分析识别可以进行类平均的自相似肌球蛋白结构（类），以充分提高分辨率，用于将明确的晶体衍生原子结构构建到3D密度中，并量化结构变体的相对群体，然后可以将其 与生化平衡有关。这种集成的方法是相互交叉验证：生理学和XRD结果由EM/ET直接验证的结构模型解释，而成功捕获所需状态是通过监测力来验证的，直到时间分辨的低温捕获的时刻，通过EM/ET保留原生结构是通过EM处理的XRD监测来验证的。具体目标是通过以下方式生物化学地或机械地扰动稳态收缩以在竞争收缩模型中进行选择：1）使用EM/ET直接成像肌球蛋白结构; 2）使用快速力学和XRD探测总体平均值并交叉验证EM/ET结果; 3）确定松弛的Lethocerus粗丝的结构;以及4）将嵌合细丝结合到肌肉中以使不同肌肉响应拉伸的机制相互关联。对收缩的机制和分子理解是骨骼肌和心脏肌肉功能的综合模型的必要前提，也是理解这些机制如何在人类疾病中缺乏的必要前提，包括心脏病、肌肉肌病、肌肉损伤和肌肉减少症。 公共卫生相关性：该项目旨在通过研究肌肉运动蛋白肌球蛋白的结构动力学，了解肌肉产生力量，缩短，抵抗拉伸和做功能力的分子基础。由于所有的肌肉，包括心脏，都依赖于肌球蛋白，我们的研究结果将影响运动生理学，随着年龄增长或疾病而失去肌肉力量，以及正常肌球蛋白功能的先天性损伤，并可能导致更好的预防或治疗骨骼肌损伤或疾病，心脏病和与年龄相关的肌肉损失。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how muscles convert chemical energy into mechanical force, motion, and work. All muscles contract via the motor-protein myosin, cyclically hydrolyzing ATP and pulling on actin filaments. The individual atomic structures of myosin and actin are known, but the structural changes myosin undergoes while it is pulling on actin are still poorly understood. Our work seeks to directly visualize, at the quasi-atomic level, the full range of structural changes myosin undergoes while working. The most interesting states, i.e. when myosin is generating force, are inherently dynamic but can be cryo-trapped and studied within the sarcomere, the specifically evolved lattice of thick and thin filaments that holds myosin and actin in close proximity and high density. Our approach remains unique worldwide and integrates muscle physiology, X-ray diffraction (XRD), rapid (~1 ms) cryo-preservation, electron microscopy, 3D reconstruction using electron tomography (EM/ET), class averaging, and quasi-atomic model building using the flight muscles from Lethocerus, whose nearly crystalline filament lattice make it the best possible specimen for such structural studies. Because individual myosins are not synchronized with each other, steady-state contraction automatically contains the full ensemble of all possible myosin states reflecting the biochemical equilibrium dynamics, open to biochemical or mechanical perturbation and investigation. Muscle physiology and XRD report the functional and structural status of the dynamic ensemble average, and are complemented by rapid cryo-trapping and EM/ET that allows us to directly visualize the entire ensemble in 3D. Multivariate data analysis identifies self-similar myosin structures (classes) that can be class-averaged, to increase resolution sufficiently for building unambiguous, crystal-derived atomic structures into the 3D densities and to quantify the relative populations of structural variants, which can then be related back to the biochemical equilibrium. This integrated approach is mutually cross-validating: physiology and XRD results are interpreted by structural models that are directly validated by EM/ET, whereas successful capture of the desired state is validated by monitoring force up to the moment of time-resolved cryo-trapping, and retention of native structure by EM/ET is validated by XRD monitoring of EM processing. Specific aims are to biochemically or mechanically perturb steady-state contractions to select among competing models of contraction by: 1) using EM/ET to directly image myosin structure; 2) using rapid mechanics and XRD to probe the ensemble average and cross-validate EM/ET results; 3) determining the structure of relaxed Lethocerus thick filaments; and 4) incorporating chimeric thin filaments into muscles to cross-correlate the mechanisms by which different muscles respond to stretch. A mechanistic and molecular understanding of contraction is a necessary prerequisite for comprehensive models of muscle function, both skeletal and cardiac, and for understanding how these mechanisms are deficient in human disease, including heart disease, muscle myopathies, muscle injuries, and sarcopenia. PUBLIC HEALTH RELEVANCE: This project seeks to understand the molecular basis for muscle's capacity to generate force, shorten, resist stretch, and do work by studying the structural dynamics of muscle's motor protein, myosin. Since all muscles, including your heart, rely on myosin, our findings will bear on exercise physiology, loss of muscle strength with ageing or illness, and congenital impairments of normal myosin function, and may lead to better prevention of, or treatment for, skeletal muscle injury or disease, heart disease and age-related muscle loss.