How do ions travel across cell membranes against a gradient of concentration? The proposed experiments will provide information that will help answer this question in the specific case of the purple membrane proton pump from H. halobium. In addition, we will learn new information about the structure of a membrane bound protein, bacteriorhodopsin. The proposed experiments rest on the assumption that the purple membrane proton pump can be ultimately understood in terms of a series of proton transfer reactions across the membrane. Which amino acid side chains of bacteriorhodopsin participate in proton transfer reactions? How are the participating groups arranged in the three dimensional structure of bacteriorhodopsin? Does vitamin K participate in H+ translocation? Three main experimental approaches will be taken: 1) Amino acid sequence analysis of chemically modified bacteriorhodopsin, 2) fluorescence spectroscopy of labeled purple membrane, and 3) reconstitution of bacteriorhodopsin and vitamin K in liposomes. We will locate the sequence positions in bacteriorhodopsin of the sites affected by several reactions, including two different carbodiimides. Some of these sites appear to be involved in the proton pump mechanism. We will use fluorescence quenching and polarization techniques to study the three dimensional structure of bacteriorhodopsin and its motion during proton pumping. The amount of vitamin K in the purple membrane will be redetermined, and its role in proton pumping will be tested in liposomes. The results will help explain the mechanism of action of the simplest ion pump. Ion pumps are responsible for maintenance of cell electrolyte balance, cell energy coupling, transport of substrates, and many other membrane-mediated processes.
