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: 7432503
• 负责人: Jared Clinton Cochran
• 金额: $ 4.96万
• 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/7432503
• 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发挥作用的理解存在相当大的差距，因为缺乏运动-丝相互作用的高分辨率结构。肌动球蛋白系统不同成分的结构特征将有助于深入了解肌球蛋白马达产生力的机制，并将揭示马达结合时肌动蛋白丝内发生的任何构象变化。由于结晶所需的高浓度野生型肌动蛋白聚合存在相关问题，我将采用一种基于两个只能在一端寡聚的互补突变肌动蛋白单体的新型分子方法。通过结合这些纯化的突变肌动蛋白，我将能够分离、结晶和解析丝状肌动蛋白成核复合物（肌动蛋白三聚体或四聚体）的结构。我将通过调整纯化和结晶条件来提高来自盘基网柄菌的两个非常规肌球蛋白运动域MyoD（l级）和MyoJ（V级）的现有晶体的质量，然后解析X射线结构。最后，我将使用 X 射线晶体学、分析超速离心和凝胶过滤以及等温滴定量热法来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，肌动球蛋白系统提供适当肌肉收缩所需的力量。许多疾病，例如心脏病和肌肉营养不良，都是由肌动球蛋白复合物功能异常引起的。在非肌肉细胞中，肌动球蛋白系统对于许多重要的生物功能至关重要，包括细胞运动和细胞分裂。癌症的持续存在依赖于细胞利用肌动球蛋白机制为这些重要的生物过程提供动力的能力。这项研究将为基于结构的化疗药物设计奠定基础，这些药物可以破坏这一必要机器的功能并防止癌细胞转移。