The epsilon subunit of the F(1)F(0) complex of Escherichia coli. cross-linking studies show the same structure in situ as when isolated.
Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C). All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates.
Schulenberg
B
B
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
Capaldi
R A
RA
D000227:Adenine Nucleotides / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000227:Adenine Nucleotides; D003432:Cross-Linking Reagents; D014357:Trypsin; D006180:Proton-Translocating ATPases