title: The region from phenylalanine-28 to lysine-50 of a yeast mitochondrial ATPase inhibitor (IF1) forms an α-helix in solution.
abstract: A mitochondrial ATPase inhibitor, IF1, is a 63 amino acid residue protein that regulates the activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we constructed mutant IF1 proteins with proline residues inserted into a wide range of their primary structures to determine the location and function of α-helix in the protein. A total of 11 yeast IF1 protein mutants were expressed and purified. Proline insertions in the region 28-50 reduced α-helical contents, indicating that the region formed a helix in solution. Oligomer formation of proline mutants at the C-terminal 38-60 region was markedly reduced, indicating that the region is required for oligomerization of the protein. Proline mutants at the N-terminal 18-39 region did not inhibit F(1)F(o)-ATPase, indicating that the region is required for ATPase inhibitory activity. Inhibition of a proline insertion mutant between residues 44 and 45 that lost a large portion of the α-helix was slower, although the maximal inhibition level of the mutant protein was comparable to that of wild-type IF1. The results suggest that the helix of yeast IF1 facilitates binding to F(1) by promoting initial interaction of the proteins.
authors.affiliation: Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka, 558-8585, Japan.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka, 558-8585, Japan.
authors.forename: Naomi
authors.initials: N
authors.lastname: Nakamae
authors.affiliation: Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka, 558-8585, Japan. ichikawa@life.osaka-cu.ac.jp.
authors.forename: Naoki
authors.initials: N
authors.lastname: Ichikawa
mesh_terms: D006801:Humans; D008239:Lysine / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*
chemical_list: D010649:Phenylalanine; C412204:ATP synthase subunit 6; D025261:Mitochondrial Proton-Translocating ATPases; D008239:Lysine
keywords: ATP synthase; Alpha helix; Mitochondrial ATPase inhibitor (IF1); Proline scanning mutagenesis