Mechanisms of Polypeptide Translocation Catalyzed by Class 1 HSP100/Clp Enzymes

关于马达的两个基本问题是：1)发动机每转一圈机器能走多远；2)需要多少燃料？回答这两个问题对于理解马达如何在汽车中工作或马达如何在生物体细胞中工作至关重要。然而，对于细胞中的分子马达来说，要回答这些问题需要完全不同的技术和实验策略，因为眼睛无法直接观察到这些问题。存在于细胞中的分子马达对于大量的代谢过程是必不可少的。例如，极端高温或暴露于环境压力下的生存取决于修复或移除受损细胞成分的分子马达。如果对这些受损的成分置之不理，它们将对细胞产生灾难性的影响。在这项工作的核心，研究人员试图回答两个提出的基本问题，即两个具有代表性的马达是如何沿着它们的轨道运动的，以及它们需要多少能量来完成这样的活动。所获得的知识对于深入了解这些马达和大量类似马达的运作方式以及它们对细胞的潜在影响非常重要。此外，该结果将允许其他人更好地提出和测试各种运动蛋白的机制，这些运动蛋白使用类似的操作原理，并处于大量生物功能的核心。对我国的研究基础设施至关重要的是培养下一代能够应用本文提出的热力学和瞬态动力学方法的科学家。这一目标将通过研究生、本科生和高中研究人员的参与来实现；这一策略在以前的国家科学基金会的支持下已经成功地实施了。在所有生物体中，蛋白质重塑、ATP依赖的蛋白质水解和蛋白质分解等反应对蛋白质组的维持至关重要。然而，这些反应中的分子水平事件仍然不清楚。本研究将通过提供ClpA、ClpAP和ClpB催化多肽易位的详细分子机制来推进知识，这三种酶分别代表了催化蛋白质重塑、ATP依赖性蛋白质水解和蛋白质分解的模式酶。此外，这些结果将推动跨领域的知识，因为各种同源AAA+酶被认为使用类似的机制来催化诸如DNA复制中的夹紧加载、微管切断、膜融合、内体的形态发生和运输等不同的反应。该项目将促进教学、培训和学习，并通过大学的ChemBridge和化学学者项目分别将高中生和本科生研究人员纳入其中。提议的活动对社会的另一个好处是，这项工作将产生在应用一系列生物物理和分子生物学技术方面受过高度训练的个人。

Two fundamental questions about any motor are: 1) how far does the machine travel per turnover of the engine and 2) how much fuel is required? Answering these two questions is essential for understanding how a motor operates in a car or how a motor operates in the cell of a living organism. However, answering such questions for a molecular motor in the cell, which cannot be directly observed by eye, requires substantially different techniques and experimental strategies. Molecular motors that exist in the cell are essential for a vast array of metabolic processes. For example, survival of extreme heat or exposure to environmental stresses depends upon molecular motors that either repair or remove damaged cellular components. If such damaged components were left unattended they would have catastrophic effects on the cell. At the core of this work the researchers seek to answer the two posed fundamental questions of how two representative motors tasked with such repair activities move along their track and how much energy they require to do so. The knowledge gained will be important for gaining a deeper understanding of how these motors and a vast array of similar motors operate and potentially the impact they have on the cell. Moreover, the results will allow others to better propose and test mechanisms for a variety of motor proteins that use similar operating principles and lie at the heart of a vast array of biological functions. Vital to the research infrastructure of our nation is training the next generation of scientists with the ability to apply the thermodynamic and transient state kinetic approaches proposed here. This goal will be achieved by involving graduate, undergraduate, and high school researchers in the work; a strategy that has been successfully invoked with previous NSF support. In all organisms reactions such as protein remodeling, ATP dependent proteolysis, and protein disaggregation are essential for proteome maintenance. However, the molecular level events in these reactions remain obscure. This research will advance knowledge by providing a detailed molecular mechanism for ClpA, ClpAP, and ClpB catalyzed polypeptide translocation, which represent model enzymes that catalyze protein remodeling, ATP dependent proteolysis, and protein disaggregation, respectively. In addition, the results will advance knowledge across fields because a variety of homologous AAA+ enzymes are thought to use similar mechanisms to catalyze such disparate reactions as clamp loading in DNA replication, microtubule severing, membrane fusion, and morphogenesis and trafficking of endosomes. This project will promote teaching, training and learning and perform outreach by incorporating high school students and undergraduate researchers through the university's ChemBridge and Chemistry Scholars programs, respectively. A further benefit of the proposed activity to society is that this work will yield individuals highly trained in the application of an array of biophysical and molecular biology techniques.