Modulation of the oligomerization state of the bovine F-1-ATPase inhibitorprotein, IF1, by pH

Bovine IF1, a basic protein of 84 amino acids, is involved in the regulatio n of the catalytic activity of the F-1 domain of ATP synthase, At pH 6.5, b ut not at basic pH values, it inhibits the ATP hydrolase activity of the en zyme. The oligomeric state of bovine LF, has been investigated at various p H values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a t etramer at pH 8, and below pH 6.5, the protein is predominantly dimeric, By covalent cross-linking, it has been found that at pH 8.0 the fragment of I F, consisting of residues 44-84 forms a dimer, whereas the fragment from re sidues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved i n the pH-sensitive interconversion between dimer and tetramer, One importan t residue in the interconversion is histidine 49, Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutan t protein is active and dimeric at all pH values investigated. It is likely from MMR studies that the inhibitor protein dimerizes by forming an antipa rallel alpha-helical coiled-coil over its C-terminal region and that at hig h pH values, where the protein is tetrameric, the inhibitory regions are ma sked. The mutation of histidine 49 to lysine is predicted to abolish coiled -coil formation over residues 32-43 preventing interaction between two dime rs, forcing the equilibrium toward the dimeric state, thereby freeing the N -terminal inhibitory regions and allowing them to interact with F-1.