Reconstitution of F1-ATPase activity from Escherichia coli subunits α, β and subunit γ tagged with six histidine residues at the C-terminus

Atsuko Ekuni, Hikaru Watanabe, Nozomi Kuroda, Ken Sawada, Hiroshi Murakami, Hiroshi Kanazawa

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

An engineered γ subunit of Escherichia coli F1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag γ), respectively, was overproduced in E. coli and purified. Six histidines are included in the C-terminal extension. The reconstituted F1 containing α, β, and His-tagged γ exhibited sixty percent of the wild-type ATPase activity. The reconstituted αβHis-tag γ complex was subjected to affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin. ATPase activity was eluted specifically with imidazole. These results implied that the tag sequence protruded to the surface of the complex and did not seriously impair the activity. The reconstituted αβHis-tag γ complex, even after its binding to the resin, exhibited ATPase activity suggesting that the γ subunit, when fixed to a solid phase, may rotate the αβ complex. This system may provide a new approach for analysis of the rotation mechanisms in F1-ATPase.

Original languageEnglish
Pages (from-to)64-68
Number of pages5
JournalFEBS Letters
Volume427
Issue number1
DOIs
Publication statusPublished - May 1 1998

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Proton-Translocating ATPases
Histidine
Escherichia coli
Adenosine Triphosphatases
Resins
Affinity chromatography
Affinity Chromatography
Sepharose
Amino Acids

Keywords

  • F-ATPase
  • Fusion subunit
  • Reconstitution
  • Rotation mechanism

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Reconstitution of F1-ATPase activity from Escherichia coli subunits α, β and subunit γ tagged with six histidine residues at the C-terminus. / Ekuni, Atsuko; Watanabe, Hikaru; Kuroda, Nozomi; Sawada, Ken; Murakami, Hiroshi; Kanazawa, Hiroshi.

In: FEBS Letters, Vol. 427, No. 1, 01.05.1998, p. 64-68.

Research output: Contribution to journalArticle

Ekuni, Atsuko ; Watanabe, Hikaru ; Kuroda, Nozomi ; Sawada, Ken ; Murakami, Hiroshi ; Kanazawa, Hiroshi. / Reconstitution of F1-ATPase activity from Escherichia coli subunits α, β and subunit γ tagged with six histidine residues at the C-terminus. In: FEBS Letters. 1998 ; Vol. 427, No. 1. pp. 64-68.
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