Thin Filaments and Muscle Regulation

描述（由申请人提供）：细丝相关的肌动蛋白结合蛋白控制基于肌动球蛋白的肌肉收缩和细胞骨架形成。为了阐明肌肉细丝发挥作用所需的机制，确定所涉及的调节蛋白的结构相互作用至关重要。为了实现我们了解心脏、骨骼和平滑肌控制系统生理学的目标，我们将在基本结构水平上研究肌肉细丝的结构，并表征调节肌肉活动的细丝连接蛋白的相互作用变化。我们将使用最先进的电子显微镜和电子断层扫描结合图像分析和三维重建来建立肌动蛋白结合蛋白在细丝肌动蛋白上的大分子结构。利用这些技术：(1)我们旨在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-原肌凝蛋白调节心脏和骨骼肌活动的结构基础，这是由Ca2+结合到肌钙蛋白和肌球蛋白-过桥结合到肌动蛋白控制的。为了实现这一目标，(A)我们将通过生成单粒子和电子层析重建来测试我们新提出的肌钙蛋白-原肌球蛋白在细丝上定位的原子模型；(B)我们将检验肌钙蛋白- 1的移动结构域锁定在肌动蛋白上以限制原肌凝蛋白处于松弛肌肉的抑制“阻断”状态的假设；(C)我们将测试原肌凝蛋白假设f -肌动蛋白螺旋的轮廓为相对坚硬的螺旋状的假设，以及原肌凝蛋白是灵活的另一种观点。(2)我们将验证突变的心肌肌钙蛋白和不同的原肌凝蛋白变体通过引起原肌凝蛋白位置的不平衡而改变细丝的调节状态来扰乱肌肉调节的假设。(3)我们将评估细丝连接钙调蛋白和钙钙蛋白在确定原肌球蛋白在血管和内脏肌肉中的位置中的调节作用。我们将确定与肌动蛋白结合的星云的结构，以完成我们的细丝图。在每项研究中，f -肌动蛋白的原子分辨率图的重建将在接近原子分辨率下界定结合蛋白与肌动蛋白的分子接触（“混合晶体学”）。摘要：对肌钙蛋白-原肌球蛋白调控细丝的研究，特别是对源自心肌的正常和突变蛋白的研究，将有助于阐明心脏收缩的分子调控机制，这对追踪心血管疾病过程至关重要。对平滑肌细丝的研究将有助于理解平滑肌收缩的微调，从而揭示血管张力和肺气道阻力的关键控制，例如高血压和哮喘的决定因素。肌动蛋白丝和相关蛋白是多种细胞系统的主要参与者，强调了所提出的工作的广泛意义。我们的目标是阐明调节心血管和骨骼肌活动的控制机制。我们将在分子水平上研究由调节蛋白精心策划的结构变化，这些变化控制着肌肉的缩短和力量的产生。了解控制心肌和血管收缩和舒张的潜在分子生理学是破译心血管疾病过程、控制血压和确定药物开发新靶点的关键。

DESCRIPTION (provided by applicant): Thin filament-associated actin-binding proteins control both actomyosin-based muscle contraction and cytoskeletal formation. To elucidate the mechanisms required for muscle thin filaments to function, it is crucial to determine the structural interactions of the regulatory proteins involved. As a means of achieving our objective to understand the physiology of cardiac, skeletal and smooth muscle control systems, we will examine the architecture of muscle thin filaments at a fundamental structural level and characterize the changing interactions of thin filament-linked proteins that regulate muscle activity. We will use state-of- the-art electron microscopy and electron tomography coupled with image analysis and 3D reconstruction to establish the macromolecular structure of actin-binding proteins on thin filament actin. 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 test our newly proposed atomic model for troponin-tropomyosin localization on thin filaments by generating single particle and electron tomographic reconstructions; (B) we will test the hypothesis that mobile domains of troponin-I latch onto actin to constrain tropomyosin in the inhibitory "blocking" state characteristic of relaxed muscle; (C) we will test both the hypothesis that tropomyosin assumes the contours of the F-actin helix as a relatively stiff coiled coiled-coil and the alternative view that tropomyosin is flexible. (2) We will test the hypothesis that mutant cardiac troponin and different tropomyosin variants perturb muscle regulation by causing an imbalance in tropomyosin's position that alters the regulatory state of thin filaments. (3) We will assess the regulatory role of thin filament-linked caldesmon and calponin in defining tropomyosin position in vascular and visceral muscle. (4) We will determine the structure of nebulin bound to actin to complete our map of thin filaments. In each study, reconstructions fitted to the atomic resolution maps of F-actin will demarcate molecular contacts of binding proteins with actin at near atomic resolution ("hybrid crystallography"). Lay summary: Studies on troponin-tropomyosin regulated filaments, with particular attention devoted to normal and mutant proteins derived from cardiac muscle, will lead to an elucidation of the molecular regulatory mechanisms governing cardiac contraction, which is essential for tracing cardiovascular disease processes. Studies on smooth muscle filaments will aid in understanding the fine-tuning of smooth muscle contraction thus revealing key controls for vascular tone and pulmonary airway resistance, determinants in, e.g., hypertension and asthma. Actin filaments and associated proteins are major participates in diverse cellular systems, underscoring the broad significance of the proposed work. Our goal is to elucidate the control mechanisms that regulate cardiovascular and skeletal muscle activity. We will examine structural changes at a molecular level that are orchestrated by regulatory proteins and which control muscle shortening and force production. Understanding the underlying molecular physiology governing contraction and relaxation in heart muscle and blood vessels is key to deciphering cardiovascular disease processes, controlling blood pressure and identifying novel targets for drug development.