Structural Dynamics of Molecular Motors and the Ribosome

分子马达和核糖体的结构动力学

• 批准号: 9315836
• 负责人: YALE E GOLDMAN
• 金额: $ 45.11万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9315836
• 关键词: Active Biological Transport; Bacteria; Biochemical; Cardiovascular system; Carrier Proteins; Cell division; Cells; Cellular biology; Characteristics; Chemistry; Cystic Fibrosis; Defect; Dependence; Development; Disease; Dissociation; Duchenne muscular dystrophy; Dynein ATPase; Elongation Factor; Enhancers; Eukaryota; Fibrosis; Gene Expression; Grant; Head; Hearing; Immune response; Immunologics; In Vitro; Kinesin; Kinetics; Lead; Link; Maintenance; Mechanics; Messenger RNA; Modeling; Molecular Motors; Motion; Motor; Muscular Dystrophies; Myosin ATPase; Myosin Type I; National Institute of General Medical Sciences; Neoplasm Metastasis; Nerve Degeneration; Neurologic; Nonsense Codon; Organ; Organelles; Organism; Peptides; Pigmentation physiologic function; Play; Property; Protein Biosynthesis; Protein Isoforms; Proteins; Reaction; Regulation; Ribosomes; Role; Rotation; Signaling Molecule; Spatial Distribution; Speed; Stroke; Structure; Surface; Testing; Tissues; Total Internal Reflection Fluorescent; Vesicle Transport Pathway; arm; base; biophysical tools; cancer cell; cell motility; cellular development; conformational conversion; flexibility; mechanical force; mechanical properties; molecular dynamics; neuron development; optical traps; protein expression; protein transport; public health relevance; single molecule; single-molecule FRET; small molecule; targeted delivery; therapeutic target; tool

 DESCRIPTION (provided by applicant): Protein synthesis and active transport of vesicular cargoes are vital to development of all tissues and to the targeted delivery of organelles, proteins, and signaling molecules in eukaryotes. Accordingly, defects in protein expression and transport are linked to developmental, neurodegenerative, pigmentation, immunological, and other diseases. Knowing the detailed mechano-chemistry and structural dynamics of the ribosome and motor proteins is essential for understanding and interpreting their roles in the cell. We developed a number of powerful new biophysical tools that reveal the modulation of structural dynamics and reaction kinetics of the protein synthesis elongation cycle and molecular motors under applied mechanical force, discriminate models of energy transduction and elucidate the essential rotational motions of conformational transitions of specific domains within the elongation factors and motor proteins. We will apply these unique tools to investigate the rhythm of protein synthesis in bacteria and eukaryotes and mechanisms that functionally modulate it and the divergent biochemical and mechanical properties of myosins-I, V, VI and X, and dynein isoforms. Understanding functional properties that have that have not yet been approached at the mechanistic detail is now possible. This application combines and extends three NIGMS grants, and so has a number of aims. Our Aims in protein synthesis are to Determine the mechanisms of modulation of protein synthesis rate of 1) downstream mRNA 2o structure and 2) upstream Shine-Delgarno (SD) sequences. Aim 3) is to Develop a eukaryotic single molecule FRET platform for detailed study of peptide elongation in higher organisms. Aim 4) is to Define how read-through of premature stop codons takes place and how small molecule enhancers of read-through operate, which are promising therapies in many diseases, e.g. Duchenne muscular dystrophy and cyctic fibrosis. For molecular motors, for each of the target myosins I, V, VI and X, to 5) test the thermal search hypothesis, determine their flexibility and solve the mechanisms of their high directionality; 6) Determine the mechanical force-dependence of association of motors with their cytoskeletal tracks and the energetics of their mechanical stroke using an ultra-high-speed optical trap; 7) Determine the dynamics of ATP association and ADP and Pi dissociation from the motors under force using combined optical trapping and single molecule TIRF microscopy; 8) Determine the rotational motions of motor heads and lever arms that generate force and cargo translocation using single molecule polarized TIRF microscopy; 9) Determine motor force and force regulation on intracellular cargos, and 10) Determine how many kinesin motors are actively engaged on intracellular cargos and their spatial distribution around the cargo surface. Overall, these studies will lead to a much more general view of the mechanisms and characteristics of the ribosome and molecular motors in vitro and in live cells leading to a more rigorous understanding of their functions in cell biology and disease.

 描述（由申请人提供）：囊泡货物的蛋白质合成和主动运输对于所有组织的发育以及真核生物中细胞器、蛋白质和信号分子的靶向递送至关重要。因此，蛋白质表达和运输的缺陷与发育有关。了解核糖体和运动蛋白的详细机械化学和结构动力学对于理解和解释它们在细胞中的作用至关重要。许多强大的新生物物理工具揭示了在施加机械力下蛋白质合成延伸循环和分子马达的结构动力学和反应动力学的调节，区分能量转导模型并阐明了特定域内构象转变的基本旋转运动我们将应用这些独特的工具来研究细菌和真核生物中蛋白质合成的节律以及对其进行功能调节的机制以及肌球蛋白-I 的不同生化和机械特性， V、VI 和 X 以及动力蛋白异构体的功能特性现在已经成为可能，该应用程序结合并扩展了三项 NIGMS 资助，因此我们在蛋白质方面有许多目标。合成的目的是确定 1) 下游 mRNA 2o 结构和 2) 上游 Shine-Delgarno (SD) 序列的蛋白质合成速率调节机制。 目标 3) 是开发一种蛋白质合成速率。用于详细研究高等生物中肽延伸的真核单分子 FRET 平台 目标 4) 是定义过早终止密码子的通读是如何发生的以及通读的小分子增强剂如何运作，这是许多疾病的有希望的治疗方法。例如杜氏肌营养不良症和循环性纤维化，对于每个目标肌球蛋白 I、V、VI 和 X，测试热搜索假设，确定其灵活性并解决其高方向性的机制； 6) 使用超高速光阱确定电机与其细胞骨架轨迹的关联以及机械行程的能量学； 7) 确定电机的动力学；使用组合光学捕获和单分子 TIRF 显微镜，在力作用下 ATP 结合以及 ADP 和 Pi 与电机解离 8) 确定产生力和力的电机头和杠杆臂的旋转运动；使用单分子偏振 TIRF 显微镜进行货物易位；9) 确定细胞内货物的动力和力调节，以及 10) 确定有多少驱动蛋白马达积极参与细胞内货物及其在货物表面周围的空间分布。到 对体外和活细胞中核糖体和分子马达的机制和特征有了更全面的了解，从而可以更严格地了解它们在细胞生物学和疾病中的功能。