Reassembly of Synechocystis sp. PCC 6803 F₁‐ATPase from its over‐expressed subunits

Abstract

Subunits a,/3, and 3' of the Fl-part of cyanobacterial FoF1-ATPase have been cloned into expression vectors. Overexpressed subunit/3 was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant a and 3' subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the a and ~ subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F1-ATPase was assembled from its purified subunits a,/3, % ~ and e. The reassembled enzyme reconstituted ATP synthesis in Fl-depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.

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

“…The above‐described difficulties in folding the RrF 1 α monomer and its lower stability in comparison with the β monomer could explain the absence of any other isolated F 1 α subunits from photosynthetic sources capable of assembly into active (αβ)‐core complexes. Two earlier studies tested the appearance of ATPase activity in mixtures of recombinant α, β and γ subunits from photosynthetic sources [24,25]. Spinach urea‐solubilized subunits were refolded in presence of several chloroplast chaperons, with no possibility to isolate each refolded subunit or to assay the structure of any assembled complex [24].…”

“…Among the Synechocystis sp. PCC6803 subunits [25]β was expressed in soluble form, whereas α and γ were expressed in inclusion bodies, and no structural assays were performed after their solubilization and refolding. Although each of the isolated α and β subunits was found to bind trinitophenyl (TNP)‐ATP, their mixture gave practically no ATPase activity.…”

“…Although each of the isolated α and β subunits was found to bind trinitophenyl (TNP)‐ATP, their mixture gave practically no ATPase activity. Even the (αβγ) mixture exhibited a lower ATPase activity [25] than that obtained with an isolated native CF 1 ‐α 3 β 3 [49] or with the above‐assembled RrF 1 ‐α 1 β 1 ‐dimer (Fig. 5).…”

“…These require the development of genetic/biochemical assay systems for all three subunits including their assembly into active subcomplexes. Many systems were developed for preparation and/or assay of recombinant F 1 β subunits from various respiratory [5,14–16] and photosynthetic [17–25] sources, but very few for respiratory [14,15,26] or photosynthetic F 1 α subunits [24,25]. Furthermore, assembly of active core complexes from recombinant α and β subunits has been reported only for TF 1 α and β[27,28].…”

The coupling region of F0F1 ATP synthase: binding of the hydrophilic loop of F0 subunit c to F1

“…We have no indication that subunit γ is involved in this binding, although a concerted binding to γ and ϵ remains conceivable. Since we have expressed both recombinant F 1 subunits in E. coli [18], we can now examine binding of c sol to these proteins alone or in concert.…”