Structural Dynamics of Molecular Motors and the Ribosome

总结 蛋白质合成和囊泡货物的主动运输对于所有组织的发育和发育至关重要 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 表达和运输与发育、神经退行性、色素沉着、免疫学和 其他疾病。了解核糖体和运动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。我们开发了多项 强大的新生物物理工具，揭示蛋白质合成的结构动力学和反应动力学 应用机械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的节律和过早终止 在真核生物中。我们将阐明骨骼肌肌球蛋白的不同生化和机械特性 和非肌肉肌球蛋白 -I、V、VI 和 X。了解尚未了解的功能动力学和机制细节 以前是可以访问的，现在是可行的。该 MIRA 拨款合并了 3 项前 NIH 拨款：申请人的 细胞分子马达计划项目的一部分，向申请人提供个人 R01 资助 分子马达的生物物理机制，以及蛋白质合成方面的多项 PI 资助。所有之间的联系 这些不同的主题是它们需要通过单分子生物物理学方法进行强大的研究 它们将 P 环 NTPase 与许多常见的结构基序和原理结合在一起。他们可能低于- 通过研究和比较它们各自的结构、能量和动态特征，发挥协同作用。前- 申请正文中给出了这种协同作用的大量内容。在续订期间，我们计划 1) 继续 成功开发最先进的单分子荧光和光阱技术，2）应用 这些方法适用于一系列肌球蛋白亚型，文献中已将其描述为具有定性 不同的特性，3）构建新型细胞内力-FRET传感器用于研究机械生物学 来自细胞外周环境的信号控制细胞核中的基因表达，4) 比较和 真核蛋白质合成与细菌系统的对比机制，5）阐明详细的机制 在蛋白质合成过程中，药物对过早终止密码子（PTC）通读的增强作用 治疗 PTC 疾病（例如杜氏肌营养不良症和囊性肌营养不良症）的候选药物 纤维化) 和 5) 测试 AAA 结构域环蛋白（包括 Hsp104）进行性易位的新项目 （分解有毒的淀粉样蛋白）和剑蛋白（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和 体外和活细胞中核糖体和分子马达的特征导致更严格的研究 它们在细胞生物学和疾病中的功能的地位。

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.