Structural changes of the biological nano-machine ATP synthase storing mechanical energy used for chemical synthesis of ATP

Conversion of energy is a key process in all living organisms. The enzyme ATP synthase synthesizes ATP, the universal coin of energy in living cells, from ADP and inorganic phosphate. To carry out this endergonic reaction it uses the energy of an electro-chemical potential made up by a proton gradient across a bio-membrane. This electro-chemical energy is in a first step converted into mechanical energy: several of the enzymes subunits rotate relative to other subunits. Subsequently the mechanical energy is converted into chemical energy: the rotational movement triggers conformational changes in the nucleotide binding sites, leading to synthesis of ATP. This second energy conversion step constitutes, to our knowledge, the first known example of chemical synthesis directly driven by mechanical force. Its efficient coupling to the first energy conversion step providing the mechanical energy is crucial for ATP synthesis. However, for synthesis of 1 molecule ATP 3-4 protons successively have to cross the membrane, demanding an up to now unknown internal, spring-like energy storage mechanism. We will investigate if structural changes of subunits g and b, two parts of the enzyme that resemble a spring, are involved in intermediate energy storage during catalysis. For this purpose we will study immobilized ATP synthase in the cell membrane and in liposomes using single-molecule Foerster Resonance Energy Transfer (FRET). Structural changes within subunits g or b will be unraveled by a change of FRET efficiency between a donor and an acceptor fluorophore attached to the same subunit. The single molecule FRET experiments will be carried out with a state-of-the-art Total Internal Reflection Fluorescence Microscope (TIRFM). For this project researchers from a group experienced in molecular biology and biochemistry of ATP synthase (D. Bald) will team up with an expert group in fluorescence microspectroscopy (A. Visser). With the experimental strategy described above we expect to gain important insights not only in the mechanism of ATP synthesis, but also in the principles of biological energy conversion and storage that may be applicable to other (motor-)proteins, Furthermore, the experimental system to be established in this project will be highly useful for investigating currently debated biomedical aspects of ATP synthase. The proposed project may also give hints as how to apply principles of biological energy conversion to future nano-technological devices.