Structural Analysis of the Actomyosin System by X-ray Crystallography

X 射线晶体学对肌动球蛋白系统的结构分析

• 批准号: 7220165
• 负责人: Jared Clinton Cochran
• 金额: $ 4.68万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-12 至 2010-04-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7220165
• 关键词: Actins; Actomyosin; Behavior; Binding; Biological Process; Calorimetry; Cell division; Cells; Class; Complex; Condition; Crystallization; Dictyostelium; Dictyostelium discoideum; Disease; Enzyme Kinetics; F-Actin; Filament; Foundations; Gel Chromatography; Heart Diseases; Laboratories; Light; Malignant Neoplasms; Methodology; Microfilaments; Molecular; Motor; Muscle Cells; Muscle Contraction; Myosin ATPase; Population; Production; Research Training; Resolution; Roentgen Rays; Screening procedure; Selenomethionine; Structure; System; Techniques; Thermodynamics; Titrations; X-Ray Crystallography; analytical ultracentrifugation; base; cancer cell; cell motility; design; improved; insight; light scattering; monomer; mutant; novel; polymerization; prevent; protein function; three dimensional structure

DESCRIPTION (provided by applicant): A considerable gap in our understanding of how cytoskeletal motor proteins function is the lack of a high resolution structure of the motor-filament interaction. The structural characterization of different components of the actomyosin system will provide insight into the mechanism of force production by myosin motors and will shed light on any conformational changes that occur within the actin filament upon motor binding. Due to the problems associated with the polymerization of wild-type actin at high concentrations necessary for crystallization, I will employ a novel molecular approach based on two complementary mutant actin monomers that can only oligomerize at one end. By combining these purified mutant actins, I will be able to isolate, crystallize, and solve the structure of the filamentous actin nucleation complex (trimer or tetramer of actin). I will improve the quality of existing crystals of two unconventional myosin motor domains from Dictyostelium discoideum, MyoD (class-l) and MyoJ (class-V), by adjusting purification and crystallization conditions and then solve the X-ray structures. Finally, I will characterize the actomyosin complex using X- ray crystallography, analytical ultracentrifugation and gel filtration, and isothermal titration calorimetry to obtain a high resolution structure of the motor-filament interaction. In muscle cells, the actomyosin system provides the force necessary for proper muscle contraction. Many diseases, such as heart disease and muscular distrophy, arise from improper functioning of the actomyosin complex. In non-muscle cells, the actomyosin system is central to many important biological functions, including cell motility and cell division. The persistence of cancer relies on the cell's ability to utilize the actomyosin machinery to power these vital biological processes. This study will lay the foundation for the structure-based design of chemotheraputic agents that disrupt the function of this necessary machine and prevent the metastasis of cancer cells.

描述（由申请人提供）：我们对细胞骨架运动蛋白如何功能的理解的差距很大，这是缺乏运动丝相互作用的高分辨率结构。肌动蛋白系统的不同成分的结构表征将洞悉肌球蛋白电机的力生产机理，并将阐明运动结合后肌动蛋白丝中发生的任何构象变化。由于与结晶所需的高浓度的野生型肌动蛋白的聚合有关的问题，我将基于两种互补的突变型肌动蛋白单体的新分子方法，这些方法只能在一端降低。通过将这些纯化的突变肌动蛋白结合在一起，我将能够分离，结晶并求解丝状肌动蛋白成核复合物（肌动蛋白的三聚体或四聚体）的结构。我将通过调整纯化和结晶条件，然后求解X射线结构，从而提高来自Dictyostelium Discoideum，Myod（L）和MyoJ（class-V）的两个非常规肌球蛋白运动结构域的现有晶体质量。最后，我将使用X射线晶体学，分析性超速离心和凝胶过滤以及等温滴定量热法表征肌动球蛋白复合物，以获得运动丝相互作用的高分辨率结构。在肌肉细胞中，肌动球蛋白系统为适当的肌肉收缩提供了必要的力。许多疾病，例如心脏病和肌肉发病症，都是由于肌动球蛋白复合物的功能不当而引起的。在非肌肉细胞中，肌动球蛋白系统对于许多重要的生物学功能，包括细胞运动和细胞分裂是至关重要的。癌症的持久性取决于该细胞利用肌动球蛋白机制为这些重要的生物学过程提供动力的能力。这项研究将奠定基础化疗剂的基础，从而破坏该必要机器的功能并防止癌细胞的转移。