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

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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 study than 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.