The affinity purification and characterization of ATP synthase complexes from mitochondria.

TitleThe affinity purification and characterization of ATP synthase complexes from mitochondria.
Publication TypeJournal Article
Year of Publication2013
AuthorsRunswick, MJ, Bason, JV, Montgomery, MG, Robinson, GC, Fearnley, IM, Walker, JE
JournalOpen Biol
Volume3
Issue2
Pagination120160
Date Published2013 Feb 13
ISSN2046-2441
KeywordsAdenosine Triphosphate, Animals, ATP Synthetase Complexes, Catalysis, Cattle, Hydrolysis, Mitochondria, Models, Molecular, Protein Conformation, Protein Structure, Secondary, Protein Subunits, Proteins, Proton-Translocating ATPases, Saccharomyces cerevisiae, Sheep, Swine
Abstract

The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

DOI10.1098/rsob.120160
Alternate JournalOpen Biol
Citation Key10.1098/rsob.120160
PubMed ID23407638
PubMed Central IDPMC3603449
Grant ListMC_U105663150 / / Medical Research Council / United Kingdom