Rapid purification, reconstitution, and characterization of cysteine-less E. Cou for, ATP synthase

P. H. Kuo, R. K. Nakamoto

Research output: Contribution to journalArticle

Abstract

Cysteine-less L. coli FoKi ATP synthase was constructed by nine site-directed mutations. A total of 21 cysteinos wore substituted by alanines. The cysteinoless mutant grew on succinate as a sole carbon source, and membrane vesicles showed ATP-driven proton-pumping by quenching of acridino orange fluorescence demonstrating a functional FF] complex. The Flag-tag epitope (DYKI3DODK) was inserted directly after the N-terminal methionine of the 3 subunit to form a construct similar to that of /hou et al (Zhou, Y., Duncan, T.M., Biilygin, V.V.. Hutrheon, M.I. and Cross, R.L. (1996) Biockim. hiophys. Ac t a 1275, 96-100). The enzyme was solubilized with ,β-D-octylglucoside and purified using an M2 antibody column against the Flag epitope. Reconstitution was performed by addition of pre-formed liposomes of E. rofrlipids to the solubilized enzyme followed by dilution. Immuno-quantification of the enzyme indicated that the amount of the cysteine-less complex in membranes was approximately .I0(/ï: less than wild type, yet turnover remained similar. Arrhenius analysis of steady-state ATP hydrolysis revealed moderately increased transition state cnthalpic and entropie parameters of the membrane bound cysteine-less enzyme when compared to wild type. Those values were not significantly changed after solubilization, purification and reconstitution. These analyses indicate that the reconstituted cysteine-less enzyme is functionally similar to wild-type and provides an ideal system in which to conduct experiments requiring cysteines only at specific sites. Supported by PHS grants G M 5095 7 and GM07267.

Original languageEnglish (US)
Pages (from-to)A1448
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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