Thin Filaments and Muscle Regulation

细丝连接的肌动蛋白结合蛋白控制基于肌球蛋白的肌肉收缩和细胞骨架的形成。为了了解肌肉收缩的正常控制，表征影响肌动蛋白-肌球蛋白结合的细丝调节蛋白的结构相互作用是至关重要的。利用结构分析，我们将在基础水平上研究心肌和骨骼肌细丝的结构，并确定调节肌肉活动的细丝连接蛋白的相互作用的变化。因此，我们的主要目标是解决放松肌肉和活动肌肉中整个细丝的原子结构。我们使用最先进的电子显微镜和电子断层扫描技术。结合图像分析、三维重建(3D-EM)和计算化学，在细丝肌动蛋白上建立肌动蛋白结合蛋白的大分子结构，从而在近原子分辨率下划分结合蛋白与肌动蛋白的分子接触。使用这些技术：(1)我们的目的是通过分析原肌球蛋白和肌钙蛋白在细丝上的相互作用来确定肌钙蛋白-原肌球蛋白调节心肌和骨骼肌活动的结构基础，这种相互作用是通过钙离子与肌钙蛋白的结合和肌球蛋白与肌动蛋白的交叉桥结合来控制的。 为了实现这一目标，(A)我们将通过以越来越高的分辨率生成单颗粒和电子断层重建并通过将肌动蛋白和调控蛋白的原子分辨率晶体结构拟合到重建体积中来描述细丝上原肌球蛋白和肌钙蛋白-原肌球蛋白的完整原子结构；(B)我们将使用计算工具进一步优化这些原子结构，以最大限度地实现调控蛋白和肌动蛋白之间的化学相互作用；(C)我们将使用分子动力学协议来评估调控蛋白质的动力学以及调控状态之间可能的转变。 (2)使用同样的方法，我们将检验这一假设，即突变的心肌肌钙蛋白和原肌球蛋白通过引起不平衡的蛋白质相互作用而扰乱肌肉调节，从而改变细丝的调节状态，我们将确定这种扰动的潜在结构性原因。(3)我们将验证这样一个假设，即相邻原肌球蛋白之间的短分子重叠结构域是原肌球蛋白端到端连接所必需的，对于原肌球蛋白在细丝上形成原肌球蛋白链至关重要，并再次评估突变对这一结构的影响。(4)我们将完成对肌球蛋白结合蛋白C与细丝形成规则和周期性连接的假设的检验，细丝可能调节横纹收缩活动。

Thin filament-linked actin-binding proteins control both actomyosin-based muscle contraction and cytoskeletal formation. In order to understand the normal control of muscle contraction, it is crucial to characterize the structural interactions of thin filament regulatory proteins that influence actin-myosin association. Using structural analysis, we will examine the architecture of cardiac and skeletal muscle thin filaments at a fundamental level and determine the changing interactions of thin filament-linked proteins that regulate muscle activity. Our principal objective therefore is to solve the atomic structure of the entire thin filament in relaxed and in active muscle. We use state-of-the-art electron microscopy and electron tomography. coupled with image analysis, 3D reconstruction (3D-EM) and computational chemistry to establish the macromolecular structure of actin-binding proteins on thin filament actin and thus demarcate molecular contacts of binding proteins with actin at near atomic resolution. Using these techniques: (1) We aim to determine the structural basis of troponin-tropomyosin regulation of cardiac and skeletal muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments, which are governed by Ca2+-binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (a) we will describe the complete atomic structure of tropomyosin and troponin-tropomyosin on thin filaments by generating single particle and electron tomographic reconstructions at increasingly high resolution and by fitting atomic resolution crystal structures of actin and regulatory proteins into the reconstruction volumes; (b) we will further refine these atomic structures using computational tools to maximize chemical interactions between regulatory proteins and actin; (c) we will use Molecular Dynamics protocols to assess regulatory protein dynamics and likely transitions between regulatory states. (2) Using this same approach, we will test the hypothesis that mutant cardiac troponin and tropomyosin perturb muscle regulation by causing imbalanced protein interactions that alter the regulatory state of thin filaments, and we will determine the underlying structural reasons for such perturbations. (3) We will test the hypothesis that the short molecular overlap domain between adjacent tropomyosins, needed for end-to-end tropomyosin linkage, is vital for tropomyosin strand formation on filaments, and again assess the impact of mutants on this structure. (4) We will complete efforts to test the hypothesis that Myosin-Binding Protein C forms regular and periodic links to thin filaments, which are likely to modulate striated contractile activity.