Thin Filaments and Muscle Regulation

薄的顺磁性连接肌动蛋白结合蛋白控制肌动蛋白为基础的肌肉收缩和 细胞骨架形成为了了解肌肉收缩的正常控制， 表征影响肌动蛋白的细丝调节蛋白的结构相互作用 协会利用结构分析，我们将研究心肌和骨骼肌的结构 在基本水平上的细丝，并决定薄的顺磁性连接的相互作用的变化 调节肌肉活动的蛋白质。因此，我们的主要目标是解决原子 在放松和活动的肌肉中整个细丝的结构。我们使用最先进的 电子显微镜和电子断层摄影术。结合图像分析、三维重建（3DEM） 和计算Ehemistrv建立肌动蛋白结合蛋白的大分子结构， 细丝肌动蛋白，从而划定结合蛋白与肌动蛋白在近原子水平的分子接触 分辨率利用这些技术：（1）确定肌钙蛋白-原肌球蛋白的结构基础 通过分析原肌球蛋白和肌钙蛋白的相互作用调节心脏和骨骼肌活动 在细丝上，这是由钙离子与肌钙蛋白的结合和肌球蛋白与肌动蛋白的交联桥的结合所控制的。 为了实现这一目标，（a）我们将描述原肌球蛋白和肌钙蛋白的完整原子结构 通过产生单粒子和电子断层摄影重建在细细丝上. 越来越高的分辨率和通过拟合肌动蛋白和调节蛋白的原子分辨率晶体结构 （B）我们将使用计算工具进一步细化这些原子结构 最大化调节蛋白和肌动蛋白之间的化学相互作用;（c）我们将使用分子 动态协议，以评估调节蛋白质的动态和可能的过渡之间的监管状态。 (2)使用同样的方法，我们将检验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来扰乱肌肉调节， 细丝，我们将确定这种扰动的潜在结构原因。(3)我们将测试 假设相邻原肌球蛋白之间的短分子重叠结构域，需要端到端 原肌球蛋白连接，是至关重要的原肌球蛋白链形成的细丝，并再次评估的影响， 突变体在这个结构上。(4)我们将完成对肌球蛋白结合蛋白C 形成规则的和周期性的连接到细丝，这可能调节横纹收缩活动。

Thin filament-linked actin-binding proteins control both actomyosln-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-mvosin 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 microscopv and electron tomographv. coupled with image analysis, 3D reconstruction (3DEM) and computational ehemistrv 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 detennine 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 Ca^^-binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (a) we will describe the complete atomic structure of tropomyosin and troponintropomyosin 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.