Spectroscopic Probes of the Muscle Cytoskeleton

我们的长期目标是确定横纹肌中肌营养不良蛋白（dyys）和肌营养不良蛋白（Utr）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，所以我们正在对这些蛋白质进行第一次定向光谱探针（磷光、荧光和电子顺磁磁共振[EPR]）的应用。本研究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的病理生理部分是由于切除或突变的Dys无法与肌动蛋白适当相互作用，从而降低了肌肉细胞骨架（costamere）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，以及通过基因或蛋白质治疗（使用Dys或Utr或更小的结构）逆转它们的可能性非常重要。我们将使用Dys， Utr和肌动蛋白的探针来询问，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这些效果至关重要？溶液中游离的和与肌动蛋白结合的Dys和Utr的结构如何比较？在MDX小鼠中测试所提出的治疗结构？与以前通过x射线或电子显微镜获得的数据相比？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异构体时，这些问题的答案有什么不同？这些问题将通过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，并通过计算模拟将这些结果与晶体学和EM的结果相结合来解决

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.