PMID:10406951

Citation	Du, Z and Gromet-Elhanan, Z (1999) Refolding of recombinant alpha and beta subunits of the Rhodospirillum rubrum F(0)F(1) ATP synthase into functional monomers that reconstitute an active alpha(1)beta(1)-dimer. Eur. J. Biochem. 263:430-7
Abstract	The alpha subunit from the Rhodospirillum rubrum F(0)F(1) ATP synthase (RrF(1)alpha) 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 RrF(1)alpha was followed by measuring its ability to stimulate the restoration of ATP synthesis and hydrolysis in beta-less R. rubrum chromatophores reconstituted with pure native or recombinant RrF(1)beta [Nathanson, L. & Gromet-Elhanan, Z. (1998) J. Biol. Chem. 273, 10933-10938]. The refolding efficiency was found to increase with decreasing RrF(1)alpha concentrations and required high concentrations of MgATP, saturating approximately 60% when 50 microgram protein.mL(-1) were refolded in presence of 50 mM MgATP. Size-exclusion HPLC of such refolded RrF(1)alpha revealed a 50-60% decrease in its aggregated form and a parallel appearance of its monomeric peak. RrF(1)beta refolded under identical conditions appeared almost exclusively as a monomer. This procedure enabled the isolation of large amounts of a stable RrF(1)alpha monomer, which stimulated the restoration of ATP synthesis and hydrolysis much more efficiently than the refolded alpha mixture, and bound ATP and ADP in a Mg-dependent manner. Incubation of both RrF(1)alpha and beta monomers, which by themselves had no ATPase activity, resulted in a parallel appearance of activity and assembled alpha(1)beta(1)-dimers, but showed no formation of alpha(3)beta(3)-hexamers. The RrF(1)-alpha(1)beta(1)-ATPase activity was, however, very similar to the activity observed in isolated native chloroplast CF(1)-alpha(3)beta(3), indicating that these dimers contain only the catalytic nucleotide-binding site at their alpha/beta interface. Their inability to associate into an alpha(3)beta(3)-hexamer seems therefore to reflect a much lower stability of the noncatalytic RrF(1) alpha/beta interface.
Links	PubMed
Keywords	Adenosine Triphosphate/pharmacology; Dose-Response Relationship, Drug; Kinetics; Models, Chemical; Protein Folding; Proton-Translocating ATPases/chemistry; Recombinant Proteins/chemistry; Rhodospirillum rubrum/enzymology; Time Factors
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RHORU:ATPA	involved_in	GO:0006754: ATP biosynthetic process	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
RHORU:ATPA		GO:0006754: ATP biosynthetic process	ECO:0000314:		P		See figure 1 from paper.	complete CACAO 3330

References

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