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

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.

Helping to understand the molecular machinery of life, the overall goal of this proposal, is a necessary complement to the modern explosion in biological information represented by genome sequencing and other advances in molecular biology. Controlling the assembly of the flagellar motor with DNA nanostructures, a specific goal of this proposal, will be a major development in Synthetic Biology. People trained, techniques developed and ideas tested during the course of the project will spread into science, healthcare, industry and education, through the routes described below, enriching the scientific culture that is vital to the success of these areas of the UK economy. The long-range economic impact of this research will be in the fields of emerging nanotechnologies and medicine. These are far enough in the future to be very difficult to predict in detail, but could involve intelligent drug delivery and cellular therapeutics, and new anti-bacterial strategies.
The beneficiaries of this research and mechanisms for reaching them are categorized below.

Immediate beneficiaries:

1. Students and postdocs trained
An important impact of the project will be the training of active researchers. Since 2005, 19 students have graduated from my group with doctorates, of whom the majority have gone on to highly skilled careers in the scientific sector. The project will create further opportunities for training research students and postdocs.

2. The general public
The scientific understanding of biological molecular machines is an important example to demonstrate the power of a scientific, rational approach to explain the marvels of nature. This approach is crucial if the UK is to lead the world in the modern knowledge-based economy. Communication with the general public will be through several channels: a web-site that includes a description of the research at a level suitable for an educated layman, research forums, tours of the laboratory as part of the outreach effort of the Oxford Physics department.

Medium-term beneficiaries:

3. Science, Education and Industry

People trained, techniques developed and ideas tested during the course of the project will spread into science, industry and education, enriching the scientific culture that is vital to the success of these areas of the UK economy. An example from previous research is informative: we developed BackScattering Dark-Field (BSDF) microscopy for fast recording of bacterial flagellar rotation, and will use it in this proposal. A spin-off version of BSDF is currently under commercial development in partnership with OUI, Oxford University's technology transfer subsidiary, both as a general low-cost microscopic tool for the education and healthcare sectors, and as a specific diagnostic tool for malaria in the developing and developed world. The commercial potential of the methods developed in this proposal will be assessed and managed by the PI, with the assistance of OUI.

Long-term, beneficiaries:

4. General public via emerging biotechnologies and personal medicine

The long-range economic impact of this research will be in these fields. These are far enough in the future to be very difficult to predict in detail. Examples of possible future applications in bio-medicine that may be helped by this proposal include: future cellular medical therapies following from understanding and controlling protein exchange and assembly in large molecular machines; new ways to combat bacterial infection by disruption of flagellar chemotaxis and motility, following from understanding flagellar rotation and switching; "intelligent" drug delivery by a synthetic biological therapeutic agent with some of the capabilities of pathogenic bacteria, following from the understanding of how to assemble and control a molecular motor and molecular switch.