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 biochemi...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 2001-03, Vol.387 (2), p.180-187
Main Authors: Tomashek, John J., Poposki, Julie A., Brusilow, William S.A.
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Amino Acid Motifs - physiology
ATP synthase
ATPase
bioenergetics
Cell Membrane - enzymology
Chromatography, Affinity
Dicyclohexylcarbodiimide - pharmacology
E. coli membrane
Electrophoresis, Polyacrylamide Gel
Enzyme Activation - drug effects
Enzyme Inhibitors - pharmacology
Escherichia coli
his-tag
Histidine - chemistry
Histidine - genetics
Liposomes - chemistry
Liposomes - metabolism
Mitochondrial Proton-Translocating ATPases
Plasmids - genetics
Protein Subunits
Proton Pumps - metabolism
proton transport
Proton-Translocating ATPases - genetics
Proton-Translocating ATPases - isolation & purification
Proton-Translocating ATPases - metabolism
Spectrometry, Fluorescence
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Summary: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.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.2000.2251