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: 10166635
• 负责人: YALE E GOLDMAN
• 金额: $ 92.06万
• 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/10166635
• 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; Structure; 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.

概括 蛋白质合成和囊泡货物的主动运输对于所有组织的发展至关重要 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。了解核糖体和电机的详细机械化学和结构动力学 蛋白质对于理解和解释其在细胞中的作用至关重要。我们已经开发了许多 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 肌肉和非肌肉分子电动机的伸长周期和货物运输在应用机械下 力量。我们将应用这些独特的工具来研究蛋白质合成和过早终止的节奏 在真核生物中。我们将阐明骨骼肌肌球蛋白的发散生化和机械性能 和非肌肉肌球蛋白-I，V，VI和X。了解尚未的功能动力学和机械细节 然而，以前可以访问是可行的。这项Mira Grant合并了3个前NIH赠款：申请人的 关于细胞分子电机的程序项目的部分 分子电机的生物物理机制，以及蛋白质合成的多PI授予。所有的联系 这些不同的主题是，通过单分子生物物理学方法，它们受到强大的研究 它们将P循环NTPases与许多常见的结构基序和原理结合在一起。他们可能不足 通过研究和比较其个体结构，能量和动态特征来协同站立。前任- 该协同作用的示例是在应用程序的主体中给出的。在续签期间，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 这些方法是一系列肌球蛋白同工型，这些方法在文献中被描述为具有定性 不同的特性，3）构建一类新的细胞内力传感器来研究机械生物学 来自细胞周围环境的信号传导到控制原子核中基因表达的信号，4）比较和比较 真核蛋白质合成与细菌系统的对比机制，5）阐明了详细的机制 在蛋白质合成期间增强的nisms，过早终止密码子（PTC）通过phar-读取 是PTC疾病治疗的候选者（例如Duchenne肌肉营养不良和囊性） 纤维化）和5）一项新的企业，用于测试AAA+结构域环蛋白的过程易位，包括HSP104 （分解有毒淀粉样蛋白）和katanin（通过切断和 也与疾病有关）。总体而言，这些研究将导致对机制和 体外和活细胞中核糖体和分子电动机的特征，导致更严格的不足 它们在细胞生物学和疾病中的功能的地位。