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定义决定Dys和UtR在横纹肌中功能的分子结构和动力学，以便为了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Duchenne(DMD)和Becker(BMD)肌营养不良。要做到这一点，传统的结构生物学方法(显微镜、结晶学)是不够的，因此我们正在进行位置定向光谱探针(磷光、荧光和电子顺磁共振[EPR])的首次应用。目前的建议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的病理生理学部分是由于Dys的消融或突变未能与肌动蛋白适当地相互作用，降低了肌肉细胞骨架(腔体)的弹性，结构动力学的直接光谱检测揭示了这一点。我们认为，这些复合体的结构和动力学对于理解DMD和BMD的病理生理学以及它们可能被基因或蛋白质疗法逆转(使用Dys或UTR或更小的结构)是重要的。我们将使用Dys、UtR和肌动蛋白的探针，来问，Dys和UtR是如何影响肌动蛋白的结构动力学的？这些蛋白质的哪些片段对这些效应至关重要？染料和非编码区在溶液中的结构如何，游离的和与肌动蛋白结合的，相互比较？建议的治疗结构在MDX小鼠身上进行测试？与之前通过X光或EM获得的结果进行比较？肌动蛋白如何影响UtR和Dys的结构动力学？导致DMD或BMD的Dys突变对这些结果有何影响？当使用肌动蛋白的G异构体时，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，使用荧光和EPR来绘制蛋白质结构和相互作用图，并通过计算模拟将这些结果与结晶学和EM的结果相结合。这个项目可能会对 了解肌肉细胞骨架，与肌肉营养不良症特别相关，因为该项目是独一无二的和及时的。我们的建议是首次对肌动蛋白-肌营养不良蛋白和肌动蛋白-滋养蛋白系统进行彻底的结构研究。这是因为两个实验室之间的创新合作--在肌肉蛋白质光谱探测方面处于世界领先地位的Thomas实验室，以及在相关蛋白质的表达和纯化方面处于世界领先地位的Erasti实验室，以及它们在小鼠模型上的生理测试。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。这项拟议的研究结果将为未来的治疗开发提供结构-功能指南。 与公共卫生相关：这个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，肌肉细胞骨架在肌肉营养不良和其他疾病中发挥着重要作用。我们将使用一种涉及分子探针、分子生物学和结构生物学的创新方法。这项工作旨在提供开发分子疗法所需的关键信息。

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.