Spectroscopic Probes of the Muscle Cytoskeleton

描述（由申请人提供）：肌肉细胞骨架的光谱探针我们的长期目标是确定确定横纹肌中肌营养不良蛋白（Dys）和肌营养不良蛋白（Utr）功能的分子结构和动力学，以便为理解这些蛋白质在肌肉功能和疾病中的作用提供急需的基础，例如杜氏肌营养不良症（DMD）和贝克尔肌营养不良症（BMD）。为了实现这一目标，传统的结构生物学方法（显微镜、晶体学）是不够的，因此我们正在对这些蛋白质进行定点光谱探针（磷光、荧光和电子顺磁共振 [EPR]）的首次应用。当前提案的重点是阐明肌动蛋白与 Dys 和 Utr 功能相互作用的结构动力学。 我们假设 DMD 和 BMD 的病理生理学部分源于消融或突变的 Dys 未能与肌动蛋白适当相互作用，从而降低了肌肉细胞骨架（肋骨）的弹性，正如结构动力学的直接光谱检测所揭示的那样。我们认为这些复合物的结构和动力学对于理解 DMD 和 BMD 的病理生理学以及使用 Dys 或 Utr 或更小的构建体通过基因或蛋白质治疗可能逆转它们很重要。我们将使用 Dys、Utr 和肌动蛋白探针来询问，Dys 和 Utr 如何影响肌动蛋白的结构动力学？这些蛋白质的哪些片段对于这些效应至关重要？溶液中游离的和与肌动蛋白结合的 Dys 和 Utr 的结构如何相互比较？所提出的治疗结构正在 mdx 小鼠中进行测试？与之前通过 X 射线或 EM 获得的那些？肌动蛋白如何影响 Utr 和 Dys 的结构动力学？导致 DMD 或 BMD 的 Dys 突变如何影响这些结果？当使用肌动蛋白 g 同工型时，这些问题的答案有何不同？这些问题将通过使用时间分辨磷光各向异性来检测旋转动力学、荧光和 EPR 来绘制蛋白质结构和相互作用以及计算模拟来将这些结果与晶体学和电子显微镜的结果相结合来解决。 该项目可能会产生重大影响 了解肌肉细胞骨架，特别是与肌营养不良症相关的细胞骨架，因为该项目是独特且及时的。我们的建议是对肌动蛋白-肌营养不良蛋白和肌动蛋白-肌营养不良蛋白系统的首次彻底的结构研究。这是可能的，因为两个实验室之间的创新合作——Thomas实验室在肌肉蛋白的光谱探针方面处于世界领先地位，而Ervasti实验室在相关蛋白质的表达和纯化及其在小鼠模型中的生理测试方面处于世界领先地位。这个项目是及时的，因为最近的工作指出了这些相互作用在疾病和治疗中的功能重要性。拟议研究的结果将为未来的治疗开发提供结构功能指南。 公共健康相关性：该项目的目标是提供对肌肉细胞骨架蛋白质的直接分子洞察，肌肉细胞骨架蛋白质在肌营养不良和其他疾病中发挥着重要作用。我们将使用涉及分子探针、分子生物学和结构生物学的创新方法。这项工作旨在提供分子疗法开发所需的关键信息。

DESCRIPTION (provided by applicant): Spectroscopic Probes of the Muscle Cytoskeleton Our long-term goal is to define the molecular structure and dynamics that determine the functions of dystrophin (Dys) and utrophin (Utr) in striated muscle, in order to provide a much needed foundation for the understanding of the roles of these proteins in muscle function and disease, such as Duchenne (DMD) and Becker (BMD) muscular dystrophies. To accomplish this, conventional methods of structural biology (microscopy, crystallography) are not sufficient, so we are carrying out the first applications of site-directed spectroscopic probes (phosphorescence, fluorescence, and electron paramagnetic magnetic resonance [EPR]) to these proteins. The focus of the current proposal is to elucidate the structural dynamics of functional interactions of actin with Dys and Utr. We hypothesize that the pathophysiology of DMD and BMD arises in part from the failure of the ablated or mutated Dys to interact appropriately with actin, reducing the resilience of the muscle cytoskeleton (costamere), as revealed by direct spectroscopic detection of structural dynamics. We propose that the structure and dynamics of these complexes are important for understanding the pathophysiology of DMD and BMD, and their possible reversal by gene or protein therapy, using Dys or Utr or smaller constructs. We will use probes on Dys, Utr, and actin, to ask, How do Dys and Utr affect the structural dynamics of actin? What segments of these proteins are crucial for these effects? How do structures of Dys and Utr in solution, free and bound to actin, compare with each other? with proposed therapeutic constructs being tested in mdx mice? with those obtained previously by xray or EM? How does actin affect the structural dynamics of Utr and Dys? How are these results affected by Dys mutations that cause DMD or BMD? How do the answers to these questions differ when the g isoform of actin is used? These questions will be addressed using time-resolved phosphorescence anisotropy to detect rotational dynamics, fluorescence and EPR to map protein structures and interactions, and computational simulation to integrate these results with those of crystallography and EM. This project is likely to have a major impact on the understanding of the muscle cytoskeleton, with particular relevance to muscular dystrophy, because the project is unique and timely. Our proposal is the first thorough structural investigation of the actin-dystrophin and actin-utrophin system. This is possible because of an innovative collaboration between two laboratories - the Thomas laboratory, which leads the world in spectroscopic probes of muscle proteins, and the Ervasti laboratory, which leads the world in the expression and purification of the relevant proteins, and their physiological testing n mouse models. This project is timely, because recent work points to the functional importance of these interactions in disease and therapy. The findings of the proposed research will provide structure-function guidelines for future therapeutic development. PUBLIC HEALTH RELEVANCE: The goal of this project is to provide direct molecular insight into the proteins of the muscle cytoskeleton, which plays a major role in muscular dystrophy and other diseases. We will use an innovative approach involving molecular probes, molecular biology, and structural biology. This work is designed to provide crucial information needed for the development of molecular therapies.