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

项目摘要 生物发动机是一种蛋白质机器，它将化学能转化为各种机械运动， 到细胞功能。噬菌体φ29基因组包装马达是最强大的生物马达之一 报道它负责将病毒基因组双链DNA（dsDNA）包装成预先形成的 蛋白质外壳（原衣壳）使用三磷酸腺苷（ATP）作为能量来源。Co-PI Guo专注于 解决了几十年来关于φ29电动机的装配和功能机制的基本问题。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在旋转过程中旋转而不是旋转。 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 单分子水平的传感和指纹识别。这些发现带来了巨大的潜力 强大的φ29电机，但要完全接受它为未来的纳米技术应用将不可避免地需要一个更 详细了解电机如何组装和操作，其中大部分才刚刚开始 阐明。关于功能性运动的化学计量和结构仍然存在重大争议 复杂性和DNA易位的机制。本提案的总体目标是阐明 通过对噬菌体φ29双链DNA包装马达的鉴定， 使用最先进的低温电子技术表征各种功能状态下的运动复合体 显微镜（EM）和分子动力学（MD）模拟，并利用为设计收集的知识 以及制造生物活性ATP驱动的无前帽蛋白纳米粒子， 功能.我们提出了三个具体目标，以解决这些具有挑战性的问题，通过整合先进的低温- EM（co-PI Mao）和计算（PI Cheng）方法与成熟的生物化学/分子生物学 协议（合作PI郭），以增加成功的机会和影响的结果。在目标1中，我们将联合收割机 cryo-EM和我们先进的计算图像分析技术，以识别和表征各种 运动中间体在原位。为了实现高分辨率，我们将探索控制 电机，以获得结构更均匀的标本。在目标2中，我们将实施和利用 MD模拟将Aim 1中获得的各种cryo-EM结构映射到 运动复合体，并将它们连接成分子“电影”，以阐明在原子水平上如何逐步， 运动复合体中的分布式构象动力学被协调以驱动DNA移位。终于在 目标3，我们将利用从目标1&2中收集到的对电机组装和操作的分子见解， 通过将其重组到脂质双层中来构建生物活性的无前帽蛋白的φ29模拟纳米颗粒 平台，这将为纳米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.