Structural Dynamics of Molecular Motors and the Ribosome

总结 蛋白质合成和囊泡物质的主动运输对所有组织的发育和细胞的生长至关重要。 真核生物中细胞器、蛋白质和信号分子的靶向递送。因此，蛋白质缺陷 表达和转运与发育、神经变性、色素沉着、免疫学和 其它疾病了解核糖体和马达的详细机械化学和结构动力学 蛋白质对于理解和解释它们在细胞中的作用至关重要。我们开发了一系列 强大的新生物物理工具，揭示了蛋白质合成的结构动力学和反应动力学 应用机械力作用下肌肉和非肌肉分子马达的伸长周期和货物运输 力我们将应用这些独特的工具来研究蛋白质合成和提前终止的节律 在真核生物中。我们将阐明骨骼肌肌球蛋白不同的生化和机械特性 和非肌肌球蛋白-I、V、VI和X。理解功能动力学和机械细节， 但以前可获得的现在是可行的。这MIRA补助合并3前NIH赠款：申请人的 一个程序项目的一部分，在细胞中的分子马达，个人R 01授予申请人的基本 分子马达的生物物理机制，以及蛋白质合成的多PI资助。所有的联系 这些不同的主题是，它们受到单分子生物物理学方法的强大研究 并且它们结合具有许多共同结构基序和原理的P-环NTPases。它们可能在- 通过研究和比较它们各自的结构特征、能量特征和动力特征，前 这种协同作用的例子在本申请的主体中给出。在续约期间，我们计划1）继续 成功开发了最先进单分子荧光和光阱技术，2）应用 这些方法用于一系列肌球蛋白同种型，这些同种型在文献中被描述为具有定性的 3）构建了一类新的胞内力-FRET传感器，用于研究机械生物学 从细胞的外周环境发出信号以控制细胞核中的基因表达，4）比较和 对比真核生物与细菌系统的蛋白质合成机制，5）阐明蛋白质合成的详细机制， 在蛋白质合成过程中，通过phar- 作为PTC疾病（例如杜氏肌营养不良和囊性纤维化）治疗的候选药物 纤维化）和5）一个新的风险，以测试进行性易位的AAA+结构域环蛋白，包括HSP 104 （分解有毒的淀粉样蛋白）和katanin（通过切断和调节微管长度） 也与疾病有关）。总的来说，这些研究将导致对机制的更普遍的看法， 体外和活细胞中核糖体和分子马达的特性导致更严格的under- 它们在细胞生物学和疾病中的作用。

Summary 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 have developed a number of powerful new biophysical tools that reveal the structural dynamics and reaction kinetics of the protein synthesis elongation cycle and cargo transport in muscle and non-muscle molecular motors under applied mechanical force. We will apply these unique tools to investigate the rhythm of protein synthesis and premature termination in eukaryotes. We will elucidate the divergent biochemical and mechanical properties of skeletal muscle myosin and non-muscle myosins-I, V, VI and X. Understanding functional dynamics and mechanistic detail that have not yet previously been accessible is now feasible. This MIRA grant coalesced 3 former NIH grants: the applicant's section of a program project on molecular motors in cells, an individual R01 grant to the applicant on basic biophysical mechanisms of molecular motors, and a multi-PI grant on protein synthesis. The links between all of these different topics are that they are subject to formidable study by single molecule biophysics approaches and they incorporate P-loop NTPases with many common structural motifs and principles. They can be under- stood synergistically by studying and comparing their individual structural, energetic and dynamic features. Ex- amples of this synergy are given in the body of the application. For the renewal period we plan to 1) continue the successful development of state-of-the-art single molecule fluorescence and optical trap technology, 2) apply these methods to a series of myosin isoforms that have been described in the literature as having qualitatively different properties, 3) build a new class of intracellular force-FRET sensors for studying mechanobiological signaling from the peripheral environment of a cell to control of gene expression in the nucleus, 4) compare and contrast mechanisms of eukaryotic protein synthesis with the bacterial system, 5) elucidate the detailed mecha- nisms for enhancement, during protein synthesis, of premature termination codon (PTC) read-through by phar- maceuticals that are candidates for therapy in PTC diseases (e.g. Duchenne muscular dystrophy and cystic fibrosis,) and 5) a new venture to test processive translocation by AAA+ domain ring proteins, including Hsp104 (which disaggregates toxic amyloid proteins) and katanin (which modulates microtubule length by severing and is also tied to diseases). 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 under- standing of their functions in cell biology and disease.