Spectroscopic Probes of the Muscle Cytoskeleton

肌肉细胞骨架的光谱探针

基本信息

批准号：
8916550
负责人：
David D Thomas
金额：
$ 34.2万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8916550
关键词：
Actins Address Affect Anisotropy Binding Collaborations Complement Complex Computer Simulation Crystallography Cytoskeleton Data Defect Detection Development Disease Duchenne muscular dystrophy Dyes Dystrophin Electrons Engineering Ensure Failure Fluorescence Fluorescence Resonance Energy Transfer Foundations Functional disorder Funding Future Genes Goals Grant Guidelines Investigation Label Laboratories Length Magnetic Resonance Maps Measurement Measures Methods Microscopy Molecular Molecular Biology Molecular Probes Molecular Structure Muscle Muscle Proteins Muscle function Muscular Dystrophies Mutagenesis Mutate Mutation Myopathy Physiological Play Point Mutation Protein Isoforms Proteins Research Roentgen Rays Role Seeds Site Skeletal Muscle Solutions Spectrum Analysis Striated Muscles Structural Models Structure System Testing Therapeutic Time Utrophin Work base design disease-causing mutation gene therapy innovation insight mdx mouse molecular dynamics mouse model novel phosphorescence programs protein structure research study resilience structural biology therapeutic development three-dimensional modeling time use tool

项目摘要

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.

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定义确定肌肉肌肉（DYS）和Utrophin（UTR）功能的分子结构和动力学，以在肌肉中为这些肌肉和肌肉功能（例如be）的角色提供基础（例如），以提供急需的基础（例如肌肉营养不良。为此，结构生物学的常规方法（显微镜，晶体学）不够，因此我们正在执行定向的光谱探针（磷光，荧光和电子顺磁性磁共振[EPR]）的首次应用。当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的病理生理学部分是由于消融或突变的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，这是由直接光谱检测结构动力学的直接光谱检测所表明的。我们建议这些复合物的结构和动力学对于理解DMD和BMD的病理生理以及使用DYS或UTR或较小构建体的基因或蛋白质治疗的逆转至关重要。我们将使用DYS，UTR和肌动蛋白上的探针来询问，DYS和UTR如何影响肌动蛋白的结构动力学？这些蛋白质的哪些段对这些作用至关重要？ DYS和UTR的结构在溶液中如何自由和肌动蛋白相比？在MDX小鼠中测试了建议的治疗构建体？与Xray或Em之前获得的那些？肌动蛋白如何影响UTR和DYS的结构动力学？这些结果如何受到导致DMD或BMD的DYS突变的影响？当使用肌动蛋白的G同工型时，这些问题的答案有何不同？这些问题将使用时间分辨的磷光各向异性解决，以检测旋转动力学，荧光和EPR以绘制蛋白质结构和相互作用以及计算模拟，以将这些结果与晶体学和EM的结果相结合。该项目可能对了解肌肉细胞骨架，与肌肉营养不良特别相关，因为该项目是独特而及时的。我们的建议是对肌动蛋白链霉素和肌动蛋白 - 嗜血蛋白系统的首次彻底结构研究。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（Thomas Laboratory），该实验室以肌肉蛋白质的光谱探针领导着世界，而ERVASTI实验室（Ervasti Laboratory）则领导着世界的表达和纯化相关蛋白质及其生理测试N小鼠模型。该项目是及时的，因为最近的工作表明这些相互作用在疾病和治疗中的功能重要性。拟议研究的发现将为未来的治疗发展提供结构功能指南。