A functional His-tagged c subunit of the Escherichia coli F-type ATPase/Synthase.
The c subunit of the Escherichia coli F0 has been tagged with a hexahistidine motif at its C-terminus. The tagged subunit is capable of forming functional F0 complexes that translocate protons in the absence of the F1 complex. In the presence of F1, the two sectors associate and display all biochemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity, ATP synthase activity, and ATP-dependent proton pumping. The enzyme can be solubilized and purified as an intact complex under native conditions on immobilized-metal affinity chromatography (IMAC) resin. The purified complex can be reincorporated into liposomes and demonstrates ATP-dependent proton pumping activity. Hexahistine tags placed at the N-terminus, in contrast, were all inactive. These experiments demonstrate the feasibility of tagging the c subunit for further studies of the F0 and suggest an important role for the N-terminus of the c subunit in either assembly or function of the protein.
Tomashek
J J
JJ
Wayne State University School of Medicine, Department of Biochemistry, Detroit, Michigan 48201, USA.
Poposki
J A
JA
Brusilow
W S
WS
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases; D010957:Plasmids / Q000235:genetics; D021122:Protein Subunits; D017494:Proton Pumps / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D008081:Liposomes; D021122:Protein Subunits; D017494:Proton Pumps; D006639:Histidine; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; C103607:mitochondrial ATPase subunit c; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases