THE DYNAMICS AND PATHOLOGIES OF MOLECULAR MOTORS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人们认识到活细胞的许多基本功能是由蛋白质复合体组成的纳米级马达完成的，这引发了人们对其机制的强烈理解。我们专注于F1 ATPase和DNA聚合酶I，这两个在生物学中具有基本重要性的非常不同的分子马达。我们建议进行原子尺度的模拟，以获得实验中无法获得的信息。这项研究将使人们更深入地了解这些马达的功能，并在DNA聚合酶的情况下，展示如何防止故障。F1 ATPase是最小的生物旋转马达，由七个单元组成，其中六个单元(α3beta3)围绕中心轴形成球状结构，伽马亚基由于催化β亚基中的ATP水解而旋转1000/秒；当施加的扭矩反向旋转g亚基时，马达合成其在细胞中的正常功能ATP。我们以前的研究探讨了构象变化的途径和机械耦合的性质。关键的下一步是评估化学步骤(ATP的水解或合成)和旋转途径上的亚基构象之间的偶联。自由能模拟将被用来寻找有利于水解或合成的构象。这种构象的存在是这一非凡马达的一个重要方面，它使ATP的有效合成或水解成为可能，这取决于g亚基的旋转方向。在给定这些构象的情况下，将进行量子力学/分子力学联合模拟来评估反应的自由能垒，并阐明催化速率提高的原因。DNA聚合酶负责将遗传信息从一个细胞世代准确复制到下一代细胞。我们之前的研究已经确定了移位步骤的细节，这是运动功能的一个重要部分。它发生在将碱基添加到引物链上之后，以便将聚合酶定位在DNA上，以添加下一个碱基。这一分析的结果将使探索DNA错配(即可能导致癌症的关键错误)阻碍DNA复制的机制成为可能，这是一种实现基本高保真的机制。已知的碱基不匹配的聚合酶I与DNA结合的晶体结构使模拟成为可能，以确定这些对易位步骤的影响。此外，在一个新的单分子实验的基础上，将探索失配对减缓手指合拢转变的影响。最后，同样使用已知的晶体结构，将执行自由能模拟，以确定失配对活性中心构型的影响。这些结果将通过提供对某些病理的理解来补充我们对正常DNA复制的分析。这具有相当重要的医学意义，同时也是人们感兴趣的问题，也是密集的实验研究的主题。两个非常不同但重要的发动机被包括在同一个提案中，因为研究的互补性将使结果更加有意义。这两个领域的研究都需要多个密切相关的模拟，这些模拟可以并行进行，效率最高。此外，由于后续计算的具体细节严重依赖于先前的结果，因此快速执行这些模拟也是至关重要的。考虑到正在研究的系统的巨大规模(F1 ATPase约为180,000个粒子，DNA聚合酶I复合体约为140,000个粒子)，一台最先进的超级计算机不仅对于遵循标准范例的大规模生产至关重要，而且作为与计算设计密切耦合的研究工具也是必不可少的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The realization that many essential functions of living cells are performed by nanoscale motors consisting of protein complexes has given rise to an intense effort to understand their mechanisms. We focus on F1 ATPase and DNA polymerase I, two very different molecular motors of fundamental importance in biology. We propose to perform atomic-scale simulations to obtain information not available from experiment. The research will lead to a deeper understanding of the function of these motors and in the case of DNA polymerase, show how malfunction is prevented. F1 ATPase, the smallest biological rotary motor, is composed of seven units, six of which (alpha3beta3) form a spherical globular construct around a central shaft, the gamma subunit, which rotates 1000/sec as a result of ATP hydrolysis in the catalytic beta subunits; when an applied torque rotates the g subunit in the reverse direction, the motor synthesizes ATP, its normal function in the cell. Our previous studies investigated the pathway of the conformational change and the nature of the mechanical coupling. The essential next step is to evaluate the coupling between the chemical steps (hydrolysis or synthesis of ATP) and the subunit conformations on the rotational pathway. Free energy simulations will be used to find the conformations that favor hydrolysis or synthesis. That such conformations exist is an essential aspect of this remarkable motor, which makes possible efficient synthesis or hydrolysis of ATP, depending on the direction of the rotation of the g subunit. Given these conformations, combined quantum mechanical/molecular mechanical simulations will be performed to evaluate the free energy barrier of the reaction and elucidate the origin of the catalytic rate enhancement. DNA polymerases are responsible for the accurate copying of genetic information from one cell generation to the next. Our previous studies have determined the details of the translocation step, an essential part of the motor function. It occurs after the addition of a base to the primer strand, so as to position the polymerase on the DNA for adding the next base. The results of this analysis will make possible exploration of the mechanism by which mismatches in DNA (i.e., critical errors that can cause cancer) stall DNA replication, a mechanism by which the essential high fidelity is achieved. Known crystal structures of the polymerase I bound to DNA with mismatched bases make possible simulations to determine the effect of these on the translocation step. In addition, based on a new single molecule experiment, the effect of mismatches on slowing the fingers closing transition will be explored. Finally, again using known crystal structures, free energy simulations will be performed to determine the effect of mismatches on the configuration of the active site. The results will complement our analysis of normal DNA replication by providing an understanding of certain pathologies. This is of considerable medical importance, as well being of interest itself and a subject of intense experimental research. Two very different, but important motors are included in the same proposal because the complementarity of the research will make the results all the more meaningful. The research in both areas requires multiple closely related simulations, which can be done most efficiently in parallel. Moreover, since specific details of subsequent calculations depend critically on the previous results, it is essential also to perform these simulations rapidly with fast turn-around. Given the large size of the systems under investigation (on the order of 180,000 particles for F1 ATPase and 140,000 for DNA polymerase I complex), a state-of-the-art supercomputer will be essential not only for large-scale production following the standard paradigm, but also as a research tool intimately coupled to the computational design.