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: 10469325
• 负责人: YALE E GOLDMAN
• 金额: $ 89.01万
• 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 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10469325
• 关键词: Active Biological Transport; Amyloid Proteins; Biochemical; Biophysical Process; Cardiovascular system; Cell Nucleus; Cell division; Cells; Cellular biology; Characteristics; Chemistry; Cystic Fibrosis; Cytoplasmic Filaments; Defect; Development; Disease; Duchenne muscular dystrophy; Environment; Eukaryota; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Grant; Hearing; Immune response; Immunologics; In Vitro; Individual; Kinetics; Lead; Length; Link; Literature; Maintenance; Methods; Microtubules; Molecular Motors; Motor; Muscle; Muscular Dystrophies; Myosin ATPase; Myosin Type I; Neoplasm Metastasis; Nerve Degeneration; Neurologic; Organ; Organelles; Peripheral; Pharmacologic Substance; Pigmentation physiologic function; Play; Property; Protein Biosynthesis; Protein Isoforms; Proteins; Reaction; Ribosomes; Role; Series; Signal Transduction; Signaling Molecule; Skeletal Muscle Myosins; System; Technology; Terminator Codon; Testing; Tissues; United States National Institutes of Health; base; biophysical techniques; biophysical tools; cancer cell; cell motility; cellular development; katanin; mechanical force; mechanical properties; molecular dynamics; neuron development; non-muscle myosin; nucleoside triphosphatase; optical traps; premature; programs; protein expression; protein transport; public health relevance; sensor; single molecule; synergism; targeted delivery; therapeutic target; tool; vesicle transport

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.

摘要 囊泡货物的蛋白质合成和主动运输对所有组织的发育和 真核生物中细胞器、蛋白质和信号分子的定向递送。因此，蛋白质中的缺陷 表达和转运与发育、神经退行性变、色素沉着、免疫学和 其他疾病。了解核糖体和马达的详细机械力化学和结构动力学 蛋白质对于理解和解释它们在细胞中的作用是必不可少的。我们已经开发了许多 揭示蛋白质合成的结构动力学和反应动力学的强大的生物物理新工具 外加机械作用下肌肉和非肌肉分子马达的伸长循环和货物运输 武力。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奏。 在真核生物中。我们将阐明骨骼肌肌球蛋白的不同生化和力学性质 和非肌肉肌球蛋白-I、V、VI和X。了解功能动力学和尚未 然而，以前是可以访问的，现在是可行的。这笔Mira补助金合并了3笔以前的NIH补助金：申请者的 关于细胞中分子马达的计划项目的一部分，个人R01在BASIC上授予申请者 分子马达的生物物理机制，以及蛋白质合成的多等电点资助。所有这些要素之间的联系 这些不同的主题是通过单分子生物物理方法进行令人敬畏的研究。 它们将P-loop NTPase与许多共同的结构主题和原则结合在一起。他们可能会被- 通过研究和比较它们各自的结构、能量和动态特征，站在了协同的立场上。前- 申请书正文中给出了这种协同效应的示例。在续约期内，我们计划1)继续 成功开发最先进的单分子荧光和光学陷阱技术，2)应用 这些方法用于一系列肌球蛋白亚型，在文献中被描述为定性地 3)构建了一类用于力学生物学研究的新型细胞内力-FRET传感器 从细胞的外围环境发出信号来控制细胞核中的基因表达，4)比较和 将真核生物蛋白质合成机制与细菌系统进行了比较，5)阐明了蛋白质合成的详细机制。 在蛋白质合成过程中，通过Phar-2增强提前终止密码子(PTC)的读出。 治疗PTC疾病(如杜氏肌营养不良和囊性肌营养不良)的候选药物 纤维化)和5)测试AAA+结构域环蛋白(包括Hsp104)进行性易位的新尝试 (它解聚有毒的淀粉样蛋白)和katanin(它通过切断和 也与疾病有关)。总体而言，这些研究将导致对这些机制和 核糖体和分子马达在体外和活细胞中的特性导致了更严格的Under- 了解它们在细胞生物学和疾病方面的功能。