Modification of subunit b of the F0 complex from Escherichia coli ATP synthase by a hydrophobic maleimide and its effects on F0 functions.
Purified F0 from Escherichia coli ATP synthase was labelled with N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide (DACM), a hydrophobic reagent which forms a stable, strongly fluorescent adduct with SH groups. Sodium dodecyl sulfate gel electrophoresis clearly demonstrated that subunit b was exclusively labelled, most likely at Cys-21, the only cysteine residue in E. coli F0. The amount of two molecules of DACM bound per F0, which was calculated from the absorption spectrum at 380 nm, is in full agreement with the postulated stoichiometry of two copies of subunit b/F0 complex. Thus the label provides a useful tool for simply detecting subunit b in protein chemical studies. DACM-labelled F0 was incorporated into liposomes and assayed for H+ translocating activity and its capacity to bind purified F1. Whereas the initial rate of H+ uptake was inhibited about 40% the reconstitution of a dicyclohexylcarbodiimide-sensitive F1F0 ATPase activity was completely unaffected. In a second set of experiments we reconstituted an F0 complex from DACM-labelled purified subunit b and an ac complex. In contrast to the results obtained with intact, DACM-labelled F0, both H+ translocating activity and F1 binding capacity were greatly reduced. Our data indicate that cysteine-21, probably together with other amino acids, is involved in maintaining a proper interaction of the hydrophobic N-terminal region of subunit b with the ac complex. This interplay seems to be a prerequisite for at least the in vitro assembly of a functional F0 complex.
Schneider
E
E
Altendorf
K
K
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D008081:Liposomes; D008301:Maleimides; D006180:Proton-Translocating ATPases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D005456:Fluorescent Dyes; D008081:Liposomes; D008301:Maleimides; D013438:Sulfhydryl Compounds; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D006180:Proton-Translocating ATPases