Explain how scientists used artificial vesicles to prove that the generation of ATP by the ATP synthase was not powered by a single high-energy intermediate but rather by a proton gradient

Question

Be sure to describe the two experiments that were negative controls (no ATP generated), the positive control (ATP generated as expected), and a fourth experiment proving that the gradient is the required energy source.

Answer 1

In all the experiments, artificial liposomes were generated and exposed to light, and the surrounding solution was checked for an increase in ATP. In the first experiment, the liposomal membranes contained only bacteriorhodopsin, a bacterial protein that pumps protons and is activated by light. In this negative control, ATP was not expected to be produced, and it was not. In the second experiment, also a negative control, the liposomes contained only ATP synthase. Again, if the chemiosmotic hypothesis was correct, ATP should not have been generated, which was what was observed. In the third experiment, both bacteriorhodopsin and ATP synthase were present in the liposomal membrane. When exposed to light, protons were pumped into the vesicle and ATP was generated. In the fourth experiment, to show that the ATP production was solely a result of the proton gradient, an uncoupling agent was added to the solution containing liposomes with bacteriorhodopsin and ATP synthase. In this case, even though the protons were being pumped into the liposomes, a gradient did not build up; this was because of the presence of the uncoupling agent, which made the membrane permeable to protons. No ATP was generated, proving that it was the proton gradient that was the energy source for ATP synthesis.