Structure, mechanism and assembly of a nano-scale biological rotary electric motor

我们现在知道，生命的基本过程是由大分子复合物完成的，它们更像机器，而不是简单的分子。21世纪科学面临的最大挑战之一就是详细地了解它们。这将对整个科学和医学产生深远的影响。在这个项目中，我建议整合对具有代表性的大型分子机器的结构和功能的理解，将这一挑战推向一个新的水平。二十年来，我一直是单分子生物学领域的先驱，该领域实时研究单个分子机器，明确地解决随机热波动问题，这从根本上区别于宏观机器。这使得我们在详细了解少数小的、相对简单的分子机器的机制方面取得了惊人的成功。下一个挑战是理解大型、复杂的分子机器。细菌鞭毛马达是一个理想的模型系统——一个直径约50纳米的旋转电动机，推动许多细菌游动。我将继续开发新的单分子技术，并利用它们来绘制鞭毛马达输入和输出之间的关系，并检测旋转和方向切换的精细结构。我对鞭毛马达中蛋白质交换的发现揭示了其结构是不断变化的，这阻碍了对其机制的发现。现在我有了理解和控制这些结构性波动的知识和实验工具。这本身就很重要；大分子机器中的蛋白质交换越来越被认为是一种重要的普遍现象。它还将为理解运动机制提供以前缺失的平台。我建议运用我独特的结构和机械经验，在前所未有的长度和时间尺度上，详细了解细菌鞭毛运动。细菌鞭毛马达是所有大分子机器中研究得最好的一个。它是许多细菌共同的旋转电动机。离子通量穿过包围细菌的细胞质膜，与转子的旋转耦合，转子跨越细菌膜和细胞壁。它通过以每秒100转的转速旋转细胞外细丝来驱动游动的细菌。由响应环境因素的信号蛋白诱导的运动方向开关，使细菌能够在营养物质和其他化学物质的梯度中导航。马达还可以作为机械传感器，对表面粘附和生物膜的形成作出决定。它对许多细菌的生活方式是不可或缺的，并且通常对生物膜的形成和毒性至关重要。马达的整体结构是已知的，它的许多组成蛋白的位置和一些组成蛋白的原子结构也是已知的。不同部件制造和组装的顺序是已知的，其功能已经在定量细节上研究了40多年。在过去的25年里，我对这一知识体系做出了重大贡献，特别是在开发生物物理工具以理解结构动力学和扭矩产生机制方面。尽管如此，结构、组装和机制的基本细节仍然不清楚，这受到我们测量的有限分辨率和结构和功能中不可预见的复杂性层的限制。这个项目的目标是实现对马达的整体结构和功能的理解，从亚毫秒的转换，动力旋转和开关，通过蛋白质交换动力学在几秒到几分钟，一直到结构的元素可能稳定几天到几个月。鞭毛马达是大型分子机器中最容易理解的例子之一，我发现的原理和方法将在其他分子机器中得到广泛的应用。

The fundamental processes of life are now known to be carried out by large molecular complexes, more like machines than simple molecules. One of the grand challenges for science in the 21st century is to understand them in detail. This will have far reaching consequences across science and medicine. In this project I propose to integrate structural and functional understanding of a representative large molecular machine, pushing this challenge to a new level. The field of Single Molecule Biology, in which I have been a pioneer for two decades, studies individual molecular machines in real time, explicitly addressing the random thermal fluctuations that distinguish them fundamentally from macroscopic machines. This has led to spectacular successes in understanding in detail the mechanisms of a handful of small, relatively simple molecular machines. The next challenge is to understand large, complicated molecular machines. The Bacterial Flagellar Motor is an ideal model system - a rotary electric motor ~50 nm in diameter that propels swimming in many bacterial species. I will continue to develop new single-molecule techniques, and use them to map the relations between flagellar motor input and output and to detect the fine-structure of rotation and directional switching. My discovery of protein exchange in the flagellar motor revealed that the structure is constantly changing, which has hindered discovery of the mechanism. Now I have the knowledge and experimental tools to understand and control these structural fluctuations. This will be significant in itself; protein exchange in large molecular machines is increasingly recognized as an important general phenomenon. It will also provide the previously-missing platform for understanding the motor mechanism. I propose to apply my unique combination of structural and mechanistic experience to understanding the bacterial flagellar motor in detail, across an unprecedented range of length and time scales. The bacterial flagellar motor is one of the best studied of all large molecular machines. It is a rotary electric motor common to many species of bacteria. Ion flux across the cytoplasmic membrane that encloses bacteria is coupled to rotation of a rotor spanning the bacterial membranes and cell wall. This drives swimming bacteria by rotating an extracellular filament at 100s of revs per second. Switches in motor direction, induced by signaling proteins in response to environmental factors, allow bacteria to navigate gradients of nutrients and other chemicals. The motor also acts as a mechanosensor, informing decisions about surface adhesion and biofilm formation. It is indispensable to the lifestyles of many bacteria, and is often crucial for biofilm formation and virulence. The overall structure of the motor is known, as are the locations of many, and atomic structures of some, of its component proteins. The order in which different parts are made and assembled is known, and its function has been studied in quantitative detail for over 4 decades. Over the last 25 years I have contributed substantially to this body of knowledge, in particular in developing biophysical tools for understanding structural dynamics and the mechanisms of torque-generation. Nonetheless, fundamental details of structure, assembly and mechanism remain unclear - constrained by the limited resolution of our measurements and by unforeseen layers of complexity in structure and function. The goal of this project is to achieve a holistic structural and functional understanding of the motor, from the sub-millisecond transitions that power rotation and switching, via protein exchange dynamics over seconds to minutes, all the way to elements of the structure that may be stable for days to months. The flagellar motor is one of the best-understood examples of a large molecular machine, and the principles and methods that I discover will find applications in a wide range of other molecular machines.