Subunit c of the F1F0 ATP synthase is a small membrane protein composed of two transmembrane segments connected by a more polar loop. Subunit c plays a central role in oxidative phosphorylation. The protein is involved in both the translocation of H+ through the F0 portion of the enzyme, and the conformational coupling of transmembrane H+ movement to ATP product release at the active sites on the F1 portion of the enzyme. A conserved carboxyl group in the middle of the C-terminal transmembrane segment (Asp61 in E. coli) is absolutely required for H+ translocation, and is thought to protonate-deprotonate during the translocation process. These changes in ionization induce the conformational change, driving release of product ATP on F1. The structure of subunit c will be determined by NMR, in both conformations which it assumes during its functional cycle. The protein has been shown to fold as a pair of interacting antiparallel helices in a mixture of chloroform-methanol-water by NMR, with the structural features predicted from a large number of biochemical and genetic experiments. The structures of the protein will be determined in this mixed solvent. Structures will also be determined in detergent micelles, for validation of those determined in the mixed solvent. The structural study of membrane proteins has been limited by the difficulty in obtaining crystals from detergent solubilized membrane proteins suitable for X-ray diffraction. The development of convenient and reliable conditions for solution structural studies will be enormously helpful in unraveling the mechanisms involved in transmembrane signaling, transport, and receptor function. A general method will be developed to assay folding of polytopic membrane proteins in mixed solvents and in detergent micelles. The folding assay involves specific 15N labeling in one transmembrane helix, with the incorporation of a nitroxide spin label in the adjacent helix. This method will be used to find optimal conditions for proper folding and for favorable NMR properties of subunit c, and potentially other membrane proteins. The folding assay will also be used, in conjunction with peptides and subunit c mutants, to analyze the structural features which stabilize the folding of membrane proteins.
