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和真核生物中细胞器、蛋白质和信号分子的定向传递至关重要。因此，蛋白质表达和运输的缺陷与发育、神经退行性变、色素沉着、免疫和其他疾病有关。了解核糖体和马达蛋白的详细机械力化学和结构动力学对于理解和解释它们在细胞中的作用是必不可少的。我们开发了许多强大的生物物理工具，揭示了蛋白质合成延伸周期和分子马达在机械力作用下的结构动力学和反应动力学的调节，区分了能量传递模型，并阐明了延伸因子和马达蛋白中特定结构域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奏和功能调节它的机制，以及肌球蛋白-I、V、VI和X以及动力蛋白亚型的不同生化和机械性质。现在有可能理解还没有在机械细节上接近的功能特性。这个应用程序结合并扩展了三个NIGMS赠款，因此有许多目标。我们在蛋白质合成方面的目标是确定1)下游mRNA2O结构和2)上游Shine-Delgarno(SD)序列对蛋白质合成速率的调控机制。目的3)建立一个真核单分子FRET平台，用于深入研究高等生物中肽的伸长。目的4)是确定过早停止密码子的通读是如何发生的，以及通读的小分子增强剂是如何起作用的，这是许多疾病的有希望的治疗方法，例如Duchenne肌营养不良和Cyctic纤维化。对于分子马达，对于每个目标肌球蛋白I、V、VI和X，5)检验热搜索假说，确定它们的灵活性，并解决它们高方向性的机制；6)使用超高速光学陷阱确定马达与其细胞骨架轨迹的结合的机械力依赖性以及它们机械行程的能量学；7)利用光学捕捉和单分子TIRF显微镜确定受力马达的ATP结合动力学以及ADP和PI的解离；8)利用单分子偏振TIRF显微镜确定马达头部和杠杆臂的旋转运动产生力和货物运输；9)确定细胞内货物上的运动力和力调节，以及10)确定细胞内货物上有多少运动蛋白马达及其在货物表面周围的空间分布。总体而言，这些研究将导致 更全面地了解核糖体和分子马达在体外和活细胞中的机制和特征，从而更严格地理解它们在细胞生物学和疾病中的功能。