Refolding of recombinant α and β subunits of the Rhodospirillum rubrum F0F1 ATP synthase into functional monomers that reconstitute an active α1β1-dimer

Ziyun Du, Zippora Gromet-Elhanan

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The α subunit from the Rhodospirillum rubrum F0F1 ATP synthase (RrF1α) was over-expressed in unc operon-deleted Escherichia coli strains under various growth conditions only in insoluble inclusion bodies. The functional refolding of urea-solubilized RrF1α was followed by measuring its ability to stimulate the restoration of ATP synthesis and hydrolysis in β-less R. rubrum chromatophores reconstituted with pure native or recombinant RrF1β [Nathanson, L. and Gromet-Elhanan, Z. (1998) J. Biol. Chem. 273, 10933-10938]. The refolding efficiency was found to increase with decreasing RrF1α concentrations and required high concentrations of MgATP, saturating ≃60% when 50 μg protein · mL-1 were refolded in presence of 50 mM MgATP. SiZe-exclusion HPLC of such refolded RrF1α revealed a 50-60% decrease in its aggregated form and a parallel appearance of its monomeric peak. RrF1β refolded under identical conditions appeared almost exclusively as a monomer. This procedure enabled the isolation of large amounts of a stable RrF1α monomer, which stimulated the restoration of ATP synthesis and hydrolysis much more efficiently than the refolded α mixture, and bound ATP and ADP in a Mg-dependent manner. Incubation of both RrF1α and β monomers, which by themselves had no ATPase activity, resulted in a parallel appearance of activity and assembled α1β1-dimers, but showed no formation of α3β3-hexamers. The RrF11β1-ATPase activity was, however, very similar to the activity observed in isolated native chloroplast CF1- α3β3, indicating that these dimers contain only the catalytic nucleotide- binding site at their crib interface. Their inability to associate into an α3β3-hexamer seems therefore to reflect a much lower stability of the noncatalytic RrF1 α/β interface.

Original languageEnglish (US)
Pages (from-to)430-437
Number of pages8
JournalEuropean Journal of Biochemistry
Volume263
Issue number2
DOIs
StatePublished - Jul 15 1999

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Rhodospirillum rubrum
Dimers
Adenosine Triphosphate
Monomers
Restoration
Adenosine Triphosphatases
Hydrolysis
Infant Equipment
Chromatophores
Inclusion Bodies
Chloroplasts
Operon
Adenosine Diphosphate
Escherichia coli
Urea
Nucleotides
Binding Sites
High Pressure Liquid Chromatography
Growth

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Refolding of recombinant α and β subunits of the Rhodospirillum rubrum F0F1 ATP synthase into functional monomers that reconstitute an active α1β1-dimer. / Du, Ziyun; Gromet-Elhanan, Zippora.

In: European Journal of Biochemistry, Vol. 263, No. 2, 15.07.1999, p. 430-437.

Research output: Contribution to journalArticle

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abstract = "The α subunit from the Rhodospirillum rubrum F0F1 ATP synthase (RrF1α) was over-expressed in unc operon-deleted Escherichia coli strains under various growth conditions only in insoluble inclusion bodies. The functional refolding of urea-solubilized RrF1α was followed by measuring its ability to stimulate the restoration of ATP synthesis and hydrolysis in β-less R. rubrum chromatophores reconstituted with pure native or recombinant RrF1β [Nathanson, L. and Gromet-Elhanan, Z. (1998) J. Biol. Chem. 273, 10933-10938]. The refolding efficiency was found to increase with decreasing RrF1α concentrations and required high concentrations of MgATP, saturating ≃60{\%} when 50 μg protein · mL-1 were refolded in presence of 50 mM MgATP. SiZe-exclusion HPLC of such refolded RrF1α revealed a 50-60{\%} decrease in its aggregated form and a parallel appearance of its monomeric peak. RrF1β refolded under identical conditions appeared almost exclusively as a monomer. This procedure enabled the isolation of large amounts of a stable RrF1α monomer, which stimulated the restoration of ATP synthesis and hydrolysis much more efficiently than the refolded α mixture, and bound ATP and ADP in a Mg-dependent manner. Incubation of both RrF1α and β monomers, which by themselves had no ATPase activity, resulted in a parallel appearance of activity and assembled α1β1-dimers, but showed no formation of α3β3-hexamers. The RrF1-α1β1-ATPase activity was, however, very similar to the activity observed in isolated native chloroplast CF1- α3β3, indicating that these dimers contain only the catalytic nucleotide- binding site at their crib interface. Their inability to associate into an α3β3-hexamer seems therefore to reflect a much lower stability of the noncatalytic RrF1 α/β interface.",
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