Structure, mechanism and assembly of a nano-scale biological rotary electric motor
Lead Research Organisation:
University of Oxford
Department Name: Oxford Physics
Abstract
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.
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.
Planned Impact
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.
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.
Publications
Armitage JP
(2020)
Assembly and Dynamics of the Bacterial Flagellum.
in Annual review of microbiology
Rieu M
(2022)
A new class of biological ion-driven rotary molecular motors with 5:2 symmetry.
in Frontiers in microbiology
Sobti M
(2020)
Cryo-EM structures provide insight into how E. coli F1Fo ATP synthase accommodates symmetry mismatch.
in Nature communications
Johnson S
(2021)
Molecular structure of the intact bacterial flagellar basal body.
in Nature microbiology
Kinosita Y
(2020)
Motile ghosts of the halophilic archaeon, Haloferax volcanii.
in Proceedings of the National Academy of Sciences of the United States of America
Kinosita Y
(2020)
Distinct chemotactic behavior in the original Escherichia coli K-12 depending on forward-and-backward swimming, not on run-tumble movements.
in Scientific reports
Afanzar O
(2021)
The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility.
in The EMBO journal
Description | We have improved 1000-fold the resolution of measurements of the rotation of biological molecular machines - by analysing the polarization of light scattered by nanometre-scale ( 1 nm = 1 billionth of a metre ) gold rods attached to nm-scale molecular motors. Rotation of the bacterial flagellar motor is delivered to the helical propeller that powers bacterial swimming via a bearing with quasi-26-fold symmetry. Our first discovery with our new resolution is that the motor pushes its way over 26 unequal barriers in a complicated interplay between the motor's stepping motion and the barriers of the molecular bearing. We have also discovered that the bearing is highly dynamic, displaying anomalous diffusion over 5 ordres of magnitude in time-scale while undregoing passive Brownian rotation. Molecular motors and bearings are on the verge of being capable of technological design and construction. Understanding how this natural example works, perfected by a billion years of evolution, will lead the way for artificial mimics. |
Exploitation Route | The gold nano-rod method will be widely used in single-molecule biology. Understanding of molecular bearings and motors will inform the design and production of synthetic mimics, which was first demonstrated in principle with rationally designed proteins since the start of this award. |
Sectors | Education Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Understanding and controlling the sub-motors of bacterial rotary nanomachines |
Amount | £217,512 (GBP) |
Funding ID | LT0039/2022-C |
Organisation | Human Frontier Science Program (HFSP) |
Sector | Charity/Non Profit |
Country | France |
Start | 09/2022 |
End | 10/2025 |
Title | CYTOP bilayer microchips with 4-microns wells of 500 nm of height |
Description | Collaborating with a company, EMAnalyticals, we designed and characterized CYTOP microchips with 4-microns wells of 500 nm of height. We were able to create lipid bilayers closing those wells, which we observed in DIC microscopy. We reproduced former experiments in the literature, injecting a fluorophore, then trapping it in the wells with a bilayer. We observed the stability of the fluorescence during several minutes after removing the fluorophore above the wells, desmonstrating the presence of the bilayer. This will serve as a basis to study the reconstituted 5:2 stators of the bacterial flagellar motor. |
Type Of Material | Technology assay or reagent |
Year Produced | 2023 |
Provided To Others? | No |
Impact | The method will allow observing and measuring the rotation of individual energized rotational 5:2 bacterial motors, nanomachines which have been recently shown to be present in several critical bacterial complexes (ExbBD, MotAB, AglRQS, TolQR) and which mechanism is still not understood. |
Title | Labelling amino acid mutation reconstitution of bacterial flagellar motor protein MotA and MotB complexes |
Description | We have successfully designed and cloned the Mot A/Mot B replace the amino acids with Cysteine to labelling and binding with bilayer. These Cysteine amino acid did not affect the rotation of flagella as we expected. We have successfully standardized using detergents to solubilize motor protein complex proteins by disrupting the lipid-protein interactions. Further we used affinity chromatography to purify the protein complexes. Currently we are using size exclusion chromatography to purify further and used it for reconstitution experiments. |
Type Of Material | Biological samples |
Year Produced | 2024 |
Provided To Others? | No |
Impact | We have successfully purified the functional motAB complex with the cysteine replacement for biotin labelling. This preliminary work opens up the possible bilayer invitro studies, including flagella rotation, by altering the proton gradients. |
Title | Labelling of the hook of bacterial flagellar motor with gold nanorods. |
Description | We developed the attachment and subsequent filtering of gold nanorods on biochemically biotinylated hooks of the bacterial flagellar motor with a large efficiency (>=1 nanorod per cell). |
Type Of Material | Technology assay or reagent |
Year Produced | 2022 |
Provided To Others? | No |
Impact | We will be able to understand the mechanism of the bacterial flagellar motor |
Title | Large FOV 2 colour 3D microscopy |
Description | We developed a Large FOV (>100um) 3D Microscopy setup which allows users to image two colours at the same time, while performing phase contrast imaging to track bacteria as they swim/glide. |
Type Of Material | Technology assay or reagent |
Year Produced | 2022 |
Provided To Others? | No |
Impact | Significant increase in data throughput. Enabled us to investigate the Flavobacteria Johnsoniae motility in gliding cells rather than fixed cells. |
Title | Membrane protein purification using chromatography techniques |
Description | We have used techniques like protein structure prediction with mutation, cloning, and bacterial rotation study. We have mainly used the Chromatography technique used to purify protein. We mainly used affinity purification and size exclusion chromatography to purify techniques using AKTA system. |
Type Of Material | Biological samples |
Year Produced | 2023 |
Provided To Others? | No |
Impact | We have successfully replaced the cysteine amino acid in motA and motB protein without affecting the flagella rotation. This suggests that protein function is not affected by this amino acid replacement. Successfully purifying this protein will help in-vitro labelling and bilayer attachment in flagella rotation study. |
Title | New strains and plasmids for the study of the bacterial flagellar motor of Escherichia Coli. |
Description | We designed four new E. Coli strains for the study of the rotation of the bacterial flagellar motor (?PAA-FliG, ?????? MotB, L55C MotB, E55C MotB) |
Type Of Material | Biological samples |
Year Produced | 2023 |
Provided To Others? | No |
Impact | We will be able to understand the mechanism of the bacterial flagellar motor |
Title | Polarization microscopy of the bacterial flagellar motor. |
Description | We developed a polarization microscope to study the rotation of the bacterial flagellar motor using the polarization of the light scattered by gold nanoantenna. |
Type Of Material | Technology assay or reagent |
Year Produced | 2022 |
Provided To Others? | No |
Impact | We have improved by 1000-fold the state-of-the-art in the product of time- and angular resolution of measurement of the orientation of nano-scale label. This will allow the mechanism of many natural and articifical molecular machines to be understood |
Title | Synthesis of liposomes encapsulating single nanorods to reconstitute membrane proteins. |
Description | Development of a lipidic nanoparticle-gold nanorod system suitable for ATP synthase incorporation to further use the single rod as label for Fo motor rotation. |
Type Of Material | Technology assay or reagent |
Year Produced | 2023 |
Provided To Others? | No |
Impact | Ability to make single molecule recordings of energized membrane proteins |
Title | - High resolution and high-speed angle data of gold nanorods labelling ATP synthase |
Description | Recording of angles of gold nanorods attached to ATP synthase encapsulated in lipid vesicles |
Type Of Material | Data analysis technique |
Year Produced | 2023 |
Provided To Others? | No |
Impact | We can measure the orientation of labels attached to energized membrane proteins. |
Title | Discrete dynamics of the undriven bacterial motor |
Description | We have collected high resolution recordings of the passive rotation of the bacterial flagellar motor (no energy source provided) and showed that it displayed unusual non-Poissonian dynamics. The transition times between most stable states are spread on a surprisingly large order of time scales, and we can detect lifetimes from a few hundred of microseconds to the a few seconds. Between those transitions, the signal displays ultra-slow diffusion, with a power-spectrum density displaying exponents smaller than one, probably a signature of a rough underlying interaction landscape, and a logarithmic mean squared displacement. We find that another property of this passive BFM dynamics is its heterogeneity, with transition rates which vary over time and state positions which display rare rearrangements. |
Type Of Material | Database/Collection of data |
Year Produced | 2023 |
Provided To Others? | No |
Impact | The data sheds light on how nature engineered imbricated protein complexes to allow a smooth rotation, a question that has been puzzling biophysicists for a long time. The anomalous diffusion that we oberve in the dataset gives insights into how one can shape energy trapping potentials so as to tune escape rates, with possible applications to the statistical physics of protein folding and protein assembly. |
Title | High resolution low load rotation of the bacterial flagellar motor of different strains of Escherichia Coli (?PAA-FliG, unplugged MotB, Chimera PotAB) |
Description | We have collected high resolution recordings of the rotation of the BFM, order of magnitude better than the current published data, using gold nanorods at a timescale of 4 microseconds and with a angular resolution of ~3°. This was accomplished by attaching 40nm gold nanorods to the hook of the BFM of different strains. We observed until now unobserved dynamic barriers in the energy landscape of the main bearing of the bacterial flagellar motor. |
Type Of Material | Data analysis technique |
Year Produced | 2023 |
Provided To Others? | No |
Impact | We now have a platform to understand the mechanism of ion-driven biological rotary motors |
Description | Flavobacteria motility imaging 3D |
Organisation | University of Oxford |
Department | Department of Biochemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Fluorescence imaging. Image analysis |
Collaborator Contribution | Strain creation. Sample preparation |
Impact | collaboration physics/biochemistry |
Start Year | 2020 |
Description | Fluorescence imaging of archaeal motor and chemotaxis proteins. |
Organisation | Albert Ludwig University of Freiburg |
Department | Faculty of Biology |
Country | Germany |
Sector | Academic/University |
PI Contribution | Fluorescence imaging of archaeal motor and chemotaxis proteins. Image analysis |
Collaborator Contribution | Produced strains. Prepared samples |
Impact | interdisciplinary biology/physics |
Start Year | 2018 |
Description | Study of 5:2 rotary motors |
Organisation | University of Copenhagen |
Department | Novo Nordisk Foundation Center for Protein Research |
Country | Denmark |
Sector | Private |
PI Contribution | Theories of the mechanism of 5:2 rotary motors |
Collaborator Contribution | Structural and bioinformatic data on the mechanism of 5:2 rotary motors |
Impact | Publication on the mechanism of 5:2 rotary motors |
Start Year | 2022 |
Description | Study of the bearing of the bacterial flagellar motor. |
Organisation | University of Montpellier |
Department | Centre for Structural Biochemistry |
Country | France |
Sector | Academic/University |
PI Contribution | Discovered interesting properties of the bearing of the bacterial flagellar motor. |
Collaborator Contribution | Produced strains. Prepared samples |
Impact | publication and further subsequent understanding of the bearing |
Start Year | 2021 |
Title | 3D Cell unrolling software |
Description | Takes 3D tracks and unrolls the cell |
Type Of Technology | Software |
Year Produced | 2021 |
Impact | N/A |
Title | 3D fluorescence tracking by back focal plane splitting software |
Description | Takes images output by the 3D tracking microscope and returns molecule tracks |
Type Of Technology | Software |
Year Produced | 2020 |
Impact | N/A |
Title | Large FOV Data Analysis |
Description | Software for the image processing and data analysis of Large FOV, 2-colour + brightfield datasets and conversion of data from lab frame of reference to cell frame of reference, with added parallelization for extra speed in processing. Also includes a GUI for more manual processing if needed. |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2023 |
Impact | Can now analyse Large FOV datasets rapidly. Will allow understanding of the mechanism of bacterial gliding |
Title | Microscopy Hardware Control |
Description | Numerous scripts in LabVIEW and Micro-Manager (bash) to control piezo stages, cameras, lasers, shutters and synchronise them via an ARDUINO board. |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2023 |
Impact | Our fluorescence microscopy systems are now more automated, making image acquisition more streamlined and faster. |
Title | PyGRod |
Description | GUI software which allows displaying and manipulating high resolution polarization data and interpreting them as rotation in the 3D space. Corrects for optical non-linearities and discrepancy in the data. Filters the data to infer relevant statistical features of the rotation. |
Type Of Technology | New/Improved Technique/Technology |
Year Produced | 2022 |
Impact | Can now treat efficiently minutes of Megahertz data and extract relevant statistical information. |