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; 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（由适用提供）：蛋白质合成和囊泡货物的主动转运对于所有组织的发展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。彼此之间，蛋白质表达和运输的缺陷与发育，神经退行性，色素沉着，免疫学和其他疾病有关。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其在细胞中的作用至关重要。我们开发了许多强大的新生物物理工具，这些工具揭示了蛋白质合成伸长循环的结构动力学和反应动力学的调节，而在施加的机械力下的分子电机，歧视能量转移模型并阐明了伸长率和运动蛋白内特定域的基本旋转运动的基本旋转运动。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以及功能调节它的机制以及肌动蛋白-I，V，VI和X和Dynein同工型的发散生化和机械性能的不同生化和机械性能。现在可以了解尚未在机械细节上接近的功能属性。该应用程序结合并扩展了三个裸体赠款，因此有许多目标。我们在蛋白质合成方面的目的是确定蛋白质合成速率调节的机理1）下游mRNA 2O结构和2）上游Shine-Delgarno（SD）序列。目的3）是开发一个真核单分子fret平台，以详细研究较高生物体的肽伸长。目的4）是定义如何进行早产密码子的读取以及如何在许多疾病中进行有前途的疗法的小分子增强子，例如Duchenne肌肉营养不良和cy骨纤维化。对于分子电动机，对于靶肌球蛋白I，V，VI和X的每个靶标电机，5）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）使用超高速度光学陷阱确定电动机与其细胞骨架轨道的缔合性和机械冲程的能量的机械力依赖性； 7）使用组合的光学诱捕和单分子TIRF显微镜确定ATP关联和ADP和PI从电动机中解离的动力学； 8）确定使用单分子极化TIRF显微镜产生力和货物易位的运动头和杠杆臂的旋转运动； 9）确定对细胞内圆柱的运动力和力调节，10）确定有多少动力蛋白电动机积极参与细胞内的碳和货物表面周围的空间分布。总体而言，这些研究将导致 在体外和活细胞中，对核糖体和分子电动机的机制和特征的一种更一般的看法，从而更加严格地了解了它们在细胞生物学和疾病中的功能。