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Powering the cell: high resolution single-molecule investigation the mechanism of ATP synthesis

Reference	BB/L01985X/1
Principal Investigator / Supervisor	Professor Richard Berry
Co-Investigators / Co-Supervisors
Institution	University of Oxford
Department	Oxford Physics
Funding type	Research
Value (£)	406,176
Status	Completed
Type	Research Grant
Start date	01/07/2014
End date	30/06/2017
Duration	36 months

Abstract

The aim of our project is to understand mechanism of ATP synthesis by F1FO, one of the most important and sophisticated of all molecular machines and the link between the primary (PMF) and secondary (ATP) forms of biological free energy. F1FO is best understood as two rotary motors F1 and FO with linked rotors and stators. The motors generate torque in opposite directions so that the overall direction of rotation depends on which motor prevails, which in turn depends on cell physiology or experimental conditions. F1 is one of the best understood of all molecular motors, but isolated FO has not been studied at a single molecule level and has not been characterized as a motor. Traditional approaches to providing a membrane environment for FO have had limited success in the context of single-molecule experiments to understand its function. We will develop two novel technologies for reconstitution of F1FO into closed, well energized lipid bilayers amenable to high numerical aperture light microscopy, and explore their use in high-resolution single molecule measurements. The first method, droplet on hydrogel bilyers, offers the prospect of full control of the membrane voltage by voltage clamping with external electrodes. The second method is based on giant unilamellar vesicles, several microns in diameter, containing a single F1FO molecule spanning the vesicle membrane. Here the PMF will be provided by pH gradients and membrane voltages derived from light-driven proton pumping by proteorhodopsin molecules in the membrane, or by the more traditional potassium valinomycin diffusion potential method. In both cases, we will use ultra-fast tracking of gold nanoparticles attached to either the rotor or stator on one side of the membrane to measure rotation, with the other part of the motor on the other side of the membrane anchored to the surface. We will use H+coupled F1FO from E. coli and, if possible, H+ and Na+coupled F1FO from various other species.

Summary

All living cells are surrounded by a cell membrane, made of a lipid bilayer two molecules thick containing among other things protein molecules that span the membrane. Proteins are the molecules that make up most of the molecular machinery that performs the basic chemical and physical processes of life. Cells form and maintain gradients of charged ions (H+ or Na+) across the membrane by pumping them across, using energy derived from sunlight or food. These gradients are used to power the cells' metabolism. Some processes use the ion gradients directly, but in most cases the cell converts their energy into a chemical form by synthesizing the high-energy molecule adenosine triphosphate (ATP) from its breakdown products adenosine diphosphate (ADP) and inorganic phosphate (Pi). A transmembrane motor protein called F1FO ATP synthase performs this conversion for most of the ATP made by most organisms. F1FO is best understood as two rotary motors, F1 and FO, with linked rotors and stators. The motors generate torque in opposite directions so that the overall direction of rotation depends on which motor generates more torque. Usually FO, which is driven by the ion gradient, is stronger and it forces F1 backwards. F1 like many other molecular machines runs on ATP, and when driven backwards it works in reverse to make ATP. This is how most ATP is synthesized. F1 is one of the best understood of all molecular motors. Isolated F1 works via a mechanism that has been revealed by two decades of groundbreaking structural and single molecule studies. Its rotor rotates counterclockwise, taking 3 steps per rev, each step coupled to the use of on molecule of ATP fuel. Experiments tracking gold particles tens of nanometers across attached to the rotor, at several thousands of video frames per second, have shown the pattern of this rotation and how it depends on the nature and state of the fuel and the motor. But isolated F1 is not found in cells. FO is much more difficult to studythan F1 because it is unstable without the membrane. A further limitation is that ATP synthesis by F1FO requires electrical separation between the opposite sides of an intact membrane and the means to produce and maintain an ion gradient. Traditional approaches to providing a membrane environment have had limited success in observing the rotation of F1FO. The aim of our project is to develop new approaches to providing a membrane environment, and to use them to understand mechanism of ATP synthesis by F1FO. We will develop two novel technologies for reconstitution of large membrane proteins into closed, well energized lipid bilayers amenable to high numerical resolution light microscopy, and explore their use in high-resolution single molecule measurements of the rotation of gold nanoparticle labels attached to fully functional F1FO and to isolated FO. We will use H+-coupled F1FO from Escherichia coli, which is a model organism, and if possible also H+- and Na+-coupled F1FO from Iliobacter tartaricus and various other bacterial species.

Impact Summary

The most significant impact of the project will be a new single-molecule research technology to study transmembrane proteins in energized lipid environment. The technology will allow simultaneous formation, perfusion, energization and optical observation of bilayers. Its immediate beneficiaries will be scientists interested in membranes and bioenergetics. Wider beneficiaries of the technology will be found in many research fields of both fundamental and commercial nature, related to processes occurring in or near biological or artificial membranes. In the long term the technology will reach the general public via its possible future application in public health and industry. Advances in ultra-fast optical nanometry and microscopy in general are likely to emerge as a component of the project. One example will be a device, currently in the early stage of patent application, for the simple and cost-effective adaptation of a commercial microscope to backscattering dark-field and interference microscopy. This device will be of a potential interest to a very broad range of users who use optical dark field microscopy. Its immediate beneficiaries will be companies related to manufacturing of optical microscopes and their components. In the long term this device may have an impact on the general public via its potential applications in medical, industrial and educational spheres. We will use the new technology to study the mechanism of ATP synthesis by F1Fo ATP synthase, a nanometre-scale biological electric turbine playing a key role in energy metabolism of all living cells. The primary impact of this part of the project will be fundamental knowledge and scientific advancement. The main beneficiaries of this research will be the international scientific community and humanity itself.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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