Refolding of recombinant alpha and beta subunits of the Rhodospirillum rubrum F0F1 ATP synthase into functional monomers that reconstitute an active alpha(1)beta(1)-dimer

The alpha subunit from the Rhodospirillum rubrum F0F1 ATP synthase (RrF(1)a lpha) was over-expressed in une operon-deleted Escherichia coli strains und er various growth conditions only in insoluble inclusion bodies, The functi onal refolding of urea-solubilized RrF(1)alpha was followed by measuring it s ability to stimulate the restoration of ATP synthesis and hydrolysis in b eta-less R. rubrum chromatophores reconstituted with pure native or recombi nant RrF(1)beta [Nathanson, L,. & Gromet-Elhanan, Z. (1998) J. Biol. Chem. 273, 10933-10938]. The refolding efficiency was found to increase with decr easing RrF(1)alpha concentrations and required high concentrations of MgATP , saturating approximate to 60% when 50 mu g protein.mL(-1) were refolded i n 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 appearanc e of its monomeric peak. RrF(1)beta refolded under identical conditions app eared almost exclusively as a monomer. This procedure enabled the isolation of large amounts of a stable RrF(1)alpha monomer, which stimulated the res toration of ATP synthesis and hydrolysis much more efficiently than the ref olded cu mixture, and bound ATP and ADP in a Mg-dependent manner. Incubatio n 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 a ctivity observed in isolated native chloroplast CF1-alpha(3)beta(3), indica ting that these dimers contain only the catalytic nucleotide-binding site a t 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 n oncatalytic RrF(1) alpha/beta interface.