Spectroscopic Probes of the Muscle Cytoskeleton

描述（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决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，如杜氏（DMD）和贝克尔（BMD）肌营养不良症。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，所以我们正在进行第一次应用定点光谱探针（磷光，荧光和电子顺磁共振[EPR]）对这些蛋白质。目前建议的重点是阐明肌动蛋白与Dys和Utr的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与肌动蛋白适当相互作用的失败，从而降低了肌肉细胞骨架（costamere）的弹性，如结构动力学的直接光谱检测所揭示的。我们建议，这些复合物的结构和动力学是很重要的了解DMD和BMD的病理生理学，以及它们可能的逆转基因或蛋白质治疗，使用Dys或UTR或更小的结构。我们将使用Dys、Utr和肌动蛋白的探针来研究Dys和Utr如何影响肌动蛋白的结构动力学？这些蛋白质的哪些部分对这些效应至关重要？Dys和Utr在溶液中的结构，游离的和与肌动蛋白结合的，如何相互比较？所提出的治疗构建体在mdx小鼠中进行测试？与以前通过X射线或EM获得的那些相比？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？当使用肌动蛋白的g亚型时，这些问题的答案有何不同？这些问题将使用时间分辨磷光各向异性来检测旋转动力学，荧光和EPR来映射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EM相结合。 该项目可能会产生重大影响， 了解肌肉细胞骨架，与肌肉萎缩症特别相关，因为该项目是独特的和及时的。我们的建议是第一次彻底的结构调查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。这是可能的，因为两个实验室之间的创新合作-托马斯实验室，领先于世界的肌肉蛋白光谱探针，和Ervasti实验室，领先于世界的表达和纯化相关蛋白质，和他们的生理测试在小鼠模型。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。 公共卫生相关性：该项目的目标是提供对肌肉细胞骨架蛋白质的直接分子见解，该蛋白质在肌肉萎缩症和其他疾病中起着重要作用。我们将使用一种创新的方法，涉及分子探针，分子生物学和结构生物学。这项工作旨在为分子疗法的发展提供所需的关键信息。

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.