Structural Dynamics of Molecular Motors and the Ribosome

 描述（由申请人提供）：蛋白质合成和囊泡货物的主动转运对于真核生物中所有组织的发育以及细胞器、蛋白质和信号分子的靶向递送至关重要。因此，蛋白质表达和转运的缺陷与发育、神经变性、色素沉着、免疫学和其他疾病有关。了解核糖体和马达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。我们开发了一些强大的新的生物物理工具，揭示了调制的结构动力学和反应动力学的蛋白质合成的延伸周期和分子马达下施加机械力，歧视模型的能量转导和阐明的基本旋转运动的特定结构域内的延伸因子和马达蛋白的构象转变。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特性。现在可以了解尚未在机械细节上接近的功能特性。这项申请结合并扩展了NIGMS的三项赠款，因此有许多目标。我们在蛋白质合成中的目标是确定1）下游mRNA 2 o结构和2）上游Shine-Delgarno（SD）序列对蛋白质合成速率的调节机制。目的3）建立真核生物单分子FRET平台，为深入研究高等生物中肽段的延伸提供基础。目的4）是定义过早终止密码子的通读如何发生以及通读的小分子增强剂如何操作，这是许多疾病（例如杜氏肌营养不良症和囊性纤维化）中有希望的疗法。对于分子马达，对于每种靶肌球蛋白I、V、VI和X，5）测试热搜索假设，确定它们的柔性并解决它们的高方向性的机制; 6）使用超高速光阱确定马达与它们的细胞骨架轨道的关联的机械力依赖性和它们的机械冲程的能量学; 7）使用组合的光学捕获和单分子TIRF显微镜确定ATP缔合以及ADP和Pi在力下从马达解离的动力学; 8）使用单分子偏振TIRF显微镜确定产生力和货物移位的马达头和杠杆臂的旋转运动; 9）确定对细胞内货物的马达力和力调节，和10）确定有多少驱动蛋白马达活跃地参与细胞内货物及其在货物表面周围的空间分布。总的来说，这些研究将导致 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认识，从而对它们在细胞生物学和疾病中的功能有了更严格的理解。

 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.