THE DYNAMICS AND PATHOLOGIES OF MOLECULAR MOTORS

分子马达的动力学和病理学

• 批准号: 7956224
• 负责人: Martin Karplus
• 金额: $ 0.08万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956224
• 关键词: ATP Hydrolysis; ATP Synthesis Pathway; Active Sites; Area; Arts; Binding; Biological; Biology; Biomedical Research; Cancer Etiology; Cells; Chemicals; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupled; Coupling; DNA; DNA Polymerase I; DNA biosynthesis; DNA-Directed DNA Polymerase; F1-ATPase; Fingers; Free Energy; Funding; Generations; Genetic; Grant; High Performance Computing; Hydrolysis; Institution; Investigation; Lead; Life; Mechanics; Medical; Molecular; Molecular Conformation; Molecular Motors; Motor; Nature; Pathology; Pathway interactions; Polymerase; Positioning Attribute; Reaction; Research; Research Personnel; Resources; Rotation; Source; Structure; System; Torque; United States National Institutes of Health; base; design; interest; large scale production; nanoscale; particle; prevent; protein complex; quantum; research study; simulation; single molecule; supercomputer; tool

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 人们认识到活细胞的许多基本功能是由蛋白质复合物组成的纳米级马达执行的，这引起了人们对了解其机制的巨大努力。我们重点关注 F1 ATP 酶和 DNA 聚合酶 I，这两种在生物学中具有根本重要性的截然不同的分子马达。我们建议进行原子尺度模拟以获得实验中无法获得的信息。这项研究将导致人们更深入地了解这些马达的功能，并以 DNA 聚合酶为例，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 (alpha3beta3) 围绕中心轴（γ 亚基）形成球形结构，由于催化 beta 中的 ATP 水解，该亚基以 1000/秒的速度旋转亚基；当施加的扭矩使 g 亚基反向旋转时，马达会合成 ATP，这是其在细胞中的正常功能。我们之前的研究调查了构象变化的途径和机械耦合的性质。下一步重要的是评估化学步骤（ATP 的水解或合成）与旋转途径上的亚基构象之间的耦合。自由能模拟将用于寻找有利于水解或合成的构象。这种构象的存在是这种非凡马达的一个重要方面，它使得 ATP 的有效合成或水解成为可能，具体取决于 g 亚基的旋转方向。考虑到这些构象，将进行量子力学/分子力学组合模拟来评估反应的自由能垒并阐明催化速率增强的根源。 DNA 聚合酶负责将遗传信息从一代细胞准确复制到下一代细胞。我们之前的研究已经确定了易位步骤的细节，这是运动功能的重要组成部分。它发生在引物链添加碱基之后，以便将聚合酶定位在DNA上以添加下一个碱基。这项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，这是实现基本高保真度的机制。聚合酶 I 与具有错配碱基的 DNA 结合的已知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，基于一项新的单分子实验，将探讨错配对减缓手指闭合转变的影响。最后，再次使用已知的晶体结构，将进行自由能模拟以确定失配对活性位点配置的影响。这些结果将通过提供对某些病理学的理解来补充我们对正常 DNA 复制的分析。这不仅具有重要的医学意义，而且本身也很有趣，也是大量实验研究的主题。两个截然不同但重要的电机包含在同一个提案中，因为研究的互补性将使结果更有意义。这两个领域的研究都需要多个密切相关的模拟，这些模拟可以最有效地并行完成。此外，由于后续计算的具体细节很大程度上取决于之前的结果，因此快速周转地快速执行这些模拟也很重要。鉴于所研究的系统规模较大（F1 ATPase 约为 180,000 个颗粒，DNA 聚合酶 I 复合物约为 140,000 个颗粒），最先进的超级计算机不仅对于遵循标准的大规模生产至关重要范式，但也作为与计算设计密切相关的研究工具。