Dissect the molecular mechanism of a viral genome packaging motor by an integrated structural approach

项目摘要 生物马达是一种蛋白质机器，它将化学能转化为各种基本的机械运动。 细胞功能。噬菌体φ29基因组包装马达是最强大的生物分子之一 据报道。它负责将病毒基因组双链DNA(DsDNA)包装成预成型的 以三磷酸腺苷(ATP)为能源的蛋白质壳(Proapsid)。联席郭皮专注于 解决了数十年来关于φ29马达的组装和功能的基本问题。最近， 郭实验室提出了一种φ29马达的旋转机制，在这种机制中，dna在 包装。他的实验室还证明了为DNA设计和改造运动通道的可行性 在单分子水平上的传感和指纹识别。这些发现带来了巨大的潜力 强大的φ29马达，但要完全接受它以用于未来的纳米技术应用，将不可避免地需要更多 对马达的装配和操作有详细的了解，其中大部分才刚刚开始。 已澄清。关于功能性马达的化学计量学和结构仍然存在重大争议 复合体和DNA转移的机制。这项提议的首要目标是澄清 噬菌体φ29dsDNA包装马达的分子机制 用最先进的冷冻电子表征发动机复合体在各种功能状态下的特性 显微镜(EM)和分子动力学(MD)模拟，并利用为设计收集的知识 并制备了一种生物活性的、由ATP驱动的无Proapsid纳米马达，它提供了前所未有的 功能性。我们提出了三个具体目标，通过整合先进的低温技术来解决这些具有挑战性的问题。 EM(co-Pi-MAO)和计算(Pi Cheng)方法与成熟的生化/分子生物学 礼仪(联合皮果)增加了成功的机会和结果的影响。在目标1中，我们将结合 Cryo-EM和我们先进的计算图像分析技术来识别和表征各种 运动中间体在原位。为了达到高分辨率，我们将探索控制动态的方法 马达以获得结构更均匀的试件。在目标2中，我们将实现和利用一个管道 MD模拟将在目标1中获得的各种低温电磁结构映射到 运动复合体，并将它们连接成分子“电影”，在原子水平上阐明如何逐步， 运动复合体中分布的构象动力学被协调以驱动DNA易位。终于来了 目标3，我们将利用从目标1和2收集的关于发动机组装和操作的分子见解来 构建具有生物活性的无前衣壳φ29模拟纳米分子 这将为纳米技术和技术的广泛应用开辟巨大的机会 纳米医学。

Project Summary Biomotors are protein machines that convert chemical energy to different kinds of mechanical motions essential to cellular functions. The bacteriophage φ29 genome packaging motor is one of the most powerful biomotors reported. It is responsible for packaging the viral genomic double-stranded DNA (dsDNA) into a preformed protein shell (procapsid) using adenosine triphosphate (ATP) as an energy source. Co-PI Guo has focused on addressing basic questions on the mechanisms of assembly and function of the φ29 motor for decades. Recently, the Guo lab proposed a revolving mechanism for the φ29 motor in which DNA revolves rather than rotates during packaging. His lab has also demonstrated the feasibility of engineering and adapting the motor channel for DNA sensing and fingerprinting at the single-molecule level. These findings bring about immense potential of the powerful φ29 motor, but to fully embrace it for future nanotechnological applications will inevitably require a more detailed understanding of how the motor assembles and operates, much of which is only beginning to be elucidated. Major controversies still exist regarding the stoichiometry and architecture of the functional motor complex and the mechanisms by which DNA is translocated. The overarching goal of this proposal is to elucidate the molecular mechanisms of the bacteriophage φ29 dsDNA packaging motor through identification and characterization of the motor complex at a variety of functional states using state-of-the-art cryo-electron microscopy (EM) and molecular dynamics (MD) simulation, and leverage the knowledge gleaned for the design and fabrication of a biologically active ATP-driven procapsid-free nanomotor that provides unprecedented functionality. We propose three specific aims to tackle these challenging problems by integrating advanced cryo- EM (co-PI Mao) and computational (PI Cheng) approaches with well-established biochemical/molecular biology protocols (co-PI Guo) to increase the chances of success and impact of the results. In Aim 1, we will combine cryo-EM and our advanced computational image analysis techniques to identify and characterize a variety of motor intermediates in situ. To achieve high resolutions, we will explore ways to control the dynamics of the motor to obtain structurally more homogeneous specimens. In Aim 2, we will implement and utilize a pipeline of MD simulations to map the various cryo-EM structures obtained in Aim 1 onto the free energy landscapes of the motor complex, and connect them into molecular “movies” to elucidate at the atomic level how stepwise, distributed conformational dynamics in the motor complex are coordinated to drive DNA translocation. Finally in Aim 3, we will leverage the molecular insights into motor assembly and operation gleaned from Aims 1&2 to construct a biologically active procapsid-free φ29 mimetic nanomotor by reconstituting it into a lipid bilayer platform, which will open up enormous opportunities for a wide range of applications in nanotechnology and nanomedicine.