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

关于任何发动机的两个基本问题是：1）发动机每转动一次，机器行驶多远; 2）需要多少燃料？弄清这两个问题对于理解汽车中的电机如何工作或电机如何在生物体的细胞中工作至关重要。然而，回答这些问题的分子马达在细胞中，这是不能直接观察到的眼睛，需要实质上不同的技术和实验策略。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，在极端高温或暴露于环境压力下的生存取决于修复或去除受损细胞成分的分子马达。如果这些受损的组件无人看管，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基本问题，即两个代表性的马达如何沿着它们的轨道沿着移动，以及它们需要多少能量来完成这些修复活动。所获得的知识对于更深入地了解这些马达和大量类似马达的工作方式以及它们对细胞的潜在影响非常重要。此外，这些结果将使其他人能够更好地提出和测试各种马达蛋白的机制，这些马达蛋白使用类似的操作原理，并处于大量生物功能的核心。对我们国家的研究基础设施至关重要的是培养下一代科学家，使他们能够应用这里提出的热力学和瞬态动力学方法。这一目标将通过让研究生、本科生和高中研究人员参与工作来实现;这一策略已在之前的NSF支持下成功实施。在所有生物体中，蛋白质重塑、ATP依赖性蛋白质水解和蛋白质解聚等反应对于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。这项研究将通过提供ClpA，ClpAP和ClpB催化的多肽易位的详细分子机制来推进知识，ClpA，ClpAP和ClpB分别代表催化蛋白质重塑，ATP依赖性蛋白水解和蛋白质解聚的模型酶。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不同的反应，如DNA复制中的钳夹加载，微管切断，膜融合以及内体的形态发生和运输。该项目将促进教学，培训和学习，并通过将高中生和本科生研究人员分别通过大学的ChemBridge和化学学者计划进行推广。拟议活动对社会的另一个好处是，这项工作将培养出在应用一系列生物物理和分子生物学技术方面训练有素的个人。

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.