Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis
1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês
10.1074/jbc.273.18.10933
ISSN1083-351X
AutoresLubov Nathanson, Zippora Gromet‐Elhanan,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoWe introduced mutations at the fully conserved residue Glu-195 in subunit β of Rhodospirillum rubrum F1-ATPase. The activities of the expressed wild type (WT) and mutant β subunits were assayed by following their capacity to assemble into the earlier prepared β-depleted, membrane-bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977)J. Biol. Chem. 252, 8742–8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, β-E195K, β-E195Q, and β-E195G, were found to bind as the WTβ into the β-depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only β-E195G could restore some of these activities when assayed in the presence of 100 mm sulfite and high MgCl2or MnCl2 concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrumF1β-E195 is equivalent to the mitochondrial F1β-E199, which points into the tunnel leading to the F1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621–628). Our findings indicate that this residue, although not an integral part of the F1catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F1 catalytic sites between different conformational states. We introduced mutations at the fully conserved residue Glu-195 in subunit β of Rhodospirillum rubrum F1-ATPase. The activities of the expressed wild type (WT) and mutant β subunits were assayed by following their capacity to assemble into the earlier prepared β-depleted, membrane-bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977)J. Biol. Chem. 252, 8742–8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, β-E195K, β-E195Q, and β-E195G, were found to bind as the WTβ into the β-depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only β-E195G could restore some of these activities when assayed in the presence of 100 mm sulfite and high MgCl2or MnCl2 concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrumF1β-E195 is equivalent to the mitochondrial F1β-E199, which points into the tunnel leading to the F1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621–628). Our findings indicate that this residue, although not an integral part of the F1catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F1 catalytic sites between different conformational states. All respiratory and photosynthetic cells contain a membrane-embedded F0F1 ATP synthase that generates ATP at the expense of the electrochemical proton gradient formed during electron transport. The catalytic F1component of the enzyme has been solubilized as a functional ATPase from many bacterial, mitochondrial, and chloroplast sources. It is a very conserved multimeric assembly with a stoichiometry of α3β3γδε and has up to six nucleotide binding sites. Three of them are catalytic sites, residing mainly on the β subunits, and three are noncatalytic, located mainly on the α subunits (1Futai M. Noumi T. Madea M. Annu. Rev. Biochem. 1989; 58: 111-136Crossref PubMed Scopus (403) Google Scholar, 2Penefsky H.S. Cross R.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 173-214PubMed Google Scholar, 3Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-839Google Scholar, 4McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 439-451Google Scholar, 5Richter M.L. Mills D.A. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 453-468Google Scholar, 6Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (392) Google Scholar). Two recently published x-ray crystallographic structures of rat liver mitochondrial MF1 at 3.6 Å (7Bianchet M. Ysern X. Hullihen J. Pedersen P.L. Amzel L.M. J. Biol. Chem. 1991; 266: 21197-21201Abstract Full Text PDF PubMed Google Scholar) and bovine heart MF1 at 2.8 Å (8Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2707) Google Scholar) have confirmed the alternate arrangement of the six large α and β subunits in a closed hexamer. The structure at 2.8 Å resolution provides direct proof for the earlier suggested location of all six nucleotide binding sites at α/β interfaces (see Ref. 9Gromet-Elhanan Z. J. Bioenerg. Biomembr. 1992; 24: 447-452Crossref PubMed Scopus (23) Google Scholar). It has also resolved two long C- and N-terminal helical domains of the γ subunit, which are embedded within the internal cavity of the α3β3hexamer, and impose on it an asymmetric structure. This asymmetry is also reflected in different conformational states displayed by the three catalytic sites on the β subunits (8Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2707) Google Scholar). The binding change mechanism (10Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (908) Google Scholar) suggested that ATP synthesis and hydrolysis involves transitions between such different conformational states via rotation of the γ-subunit relative to an α3β3subassembly. So this structural information initiated a drive to demonstrate that such rotation is possible (11Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (456) Google Scholar, 12Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (458) Google Scholar, 13Noji H. Yasuda R. Yoshida M. Kinosita K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1916) Google Scholar). These important observations do not explain how rotation drives catalytic site transitions, which must involve intricate protein-protein interactions within the α/β catalytic interfaces as well as between them and various domains on the γ subunit. Full elucidation of the unique mechanism of action of the ATP synthases will therefore require clear definition of the role of amino acid residues that are involved in catalysis, as well as identification and detailed characterization of all interacting subdomains on the α, β, and γ subunits, and their relation to the catalytic sites. Genetic/biochemical assay systems that are crucial for such studies have been developed in respiratory bacteria and yeast mitochondria (see Refs. 1Futai M. Noumi T. Madea M. Annu. Rev. Biochem. 1989; 58: 111-136Crossref PubMed Scopus (403) Google Scholar, 2Penefsky H.S. Cross R.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 173-214PubMed Google Scholar, and 6Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (392) Google Scholar), but the application of this approach to photosynthetic systems lagged behind the respiratory ones. Although the conserved structure of all isolated F1-ATPases suggests a common catalytic mechanism, there are clear differences in various properties between the respiratory and photosynthetic F0F1 and F1-complexes, for instance in regulation of activity and sensitivity to some inhibitors (3Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-839Google Scholar, 4McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 439-451Google Scholar, 5Richter M.L. Mills D.A. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 453-468Google Scholar). It is, therefore, important to develop recombinant molecular techniques and direct in vivo and/or in vitro assays of activity for photosynthetic complexes containing mutated F1-subunits. Two such systems have recently been reported for Chlamydomonas reinhardtii and spinach chloroplast F1-β subunits (14Avni A. Anderson J.D. Holland N. Rochaix J.D. Gromet-Elhanan Z. Edelman M. Science. 1992; 257: 1245-1247Crossref PubMed Scopus (59) Google Scholar, 15Hu D. Fiedler H.R. Golan T. Edelman M. Strotmann H. Shavit N. Leu S. J. Biol. Chem. 1997; 272: 5457-5463Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 16Burkovski A. Lill H. Engelbrecht S. Biochim. Biophys. Acta. 1994; 1186: 243-246Crossref PubMed Scopus (9) Google Scholar, 17Chen Z. Spies A. Hein R. Zhou X. Thomas B-C. Richter M.L. Gegenheimer P. J. Biol. Chem. 1995; 270: 17124-17132Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The F1-β subunit of the photosynthetic bacteriumRhodospirillum rubrum(RrF1β) 1The abbreviations used are: RrF1β, RrF1α, CF1β, CF1α, EcF1β, MF1β, and TF1β, β and α subunits of the F1-ATPase of R. rubrum, chloroplasts, E. coli, mitochondria, and thermophilicBacillus PS3, respectively; Bchl, bacteriochlorophyll; PCR, polymerase chain reaction; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; DCCD, dicyclohexylcarbodiimide. 1The abbreviations used are: RrF1β, RrF1α, CF1β, CF1α, EcF1β, MF1β, and TF1β, β and α subunits of the F1-ATPase of R. rubrum, chloroplasts, E. coli, mitochondria, and thermophilicBacillus PS3, respectively; Bchl, bacteriochlorophyll; PCR, polymerase chain reaction; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; DCCD, dicyclohexylcarbodiimide.provides an especially suitable system for mutational analysis, based on the available MF1 crystal structure. Thus, the published sequence of the RrF1 operon (18Falk G. Hampe A. Walker J.E. Biochem. J. 1985; 228: 391-407Crossref PubMed Scopus (94) Google Scholar) revealed that RrF1β is most closely related to MF1β. Their amino acid sequences show >76% identity, as compared with only 72% for EcF1β, 69% for tobacco CF1β (18Falk G. Hampe A. Walker J.E. Biochem. J. 1985; 228: 391-407Crossref PubMed Scopus (94) Google Scholar), and 68% for TF1β (19Ohta S. Yohda M. Ishizuka M. Hirata H. Hamamoto T. Otawara-Hamamoto Y. Matsuda K. Kagawa Y. Biochim. Biophys. Acta. 1988; 933: 141-155Crossref PubMed Scopus (119) Google Scholar). The very high similarity between RrF1β and MF1β extends also to the R. rubrum and mitochondrial F0F1 and F1 which, unlike the chloroplast and Escherichia coli enzymes, exhibit an identical sensitivity to inhibition by oligomycin and efrapeptin (20Johansson, B. C., Baltscheffsky, M., Baltscheffsky, H., Proceedings of the IInd International Congress on Photosynthetic Research, Forti, G., Avron, M., Melandri, A., 2, 1971, 1203, 1209, Dr. W. Junk N. V., The Hague, The Netherlands.Google Scholar, 21Bengis-Garber C. Gromet-Elhanan Z. Biochemistry. 1979; 18: 3577-3581Crossref PubMed Scopus (27) Google Scholar, 22Weiss S. McCarty R.E. Gromet-Elhanan Z. J. Bioenerg. Biomembr. 1994; 26: 573-581Crossref PubMed Scopus (9) Google Scholar). Furthermore, a large number ofin vitro assays of activity have been developed for the RrF1β subunit that was isolated in functional form from the chromatophore membrane-bound RrF0F1. They include the ability of the isolated RrF1β to bind ATP, ADP, and Pi (23Gromet-Elhanan Z. Kananshvili D. Biochemistry. 1984; 23: 1022-1028Crossref Scopus (39) Google Scholar, 24Khananshvili D. Gromet-Elhanan Z. Biochemistry. 1985; 24: 2482-2487Crossref Scopus (16) Google Scholar) as well as to rebind to the β-depleted enzyme and restore its lost ATP synthesis and hydrolysis activities (25Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar, 26Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar). Baltscheffsky et al. (27Baltscheffsky M. Nadanciva S. Harris D.A. Mutata N. Research in Photosynthesis. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1992: 385-388Google Scholar) have cloned the RrF1β gene and expressed it in E. coli as a fusion protein with glutathione S-transferase but did not carry out any assays of activity. We have recently cloned this gene and have expressed the RrF1β subunit in E. colilacking the whole unc operon as a soluble protein that could restore ATP synthesis to β-depleted chromatophores (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar). Here we describe the expression and full purification of RrF1β mutated in Glu-195. A detailed comparison of the activities of these mutants with those of the expressed WT β subunit revealed that RrF1β-E195 plays an important role in divalent cation-dependent ATP synthesis and hydrolysis. R. rubrum cells were grown as described previously (25Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar). Three unc operon-deleted E. coli strains, LM2800, LM3115 (29Jensen P.R. Michelsen O. J. Bacteriol. 1992; 174: 7635-7641Crossref PubMed Google Scholar), and DK8 (30Klionsky D.J. Brusilow W.S.A. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar), were used as hosts for recombinant plasmids (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar) carrying the WT and Glu-195 mutated RrF1β genes. The transformed cells were grown in LB medium supplemented with thymine (50 μg/ml), thiamine (2 μg/ml), 0.2 mm isoleucine, 0.2 mm valine, 0.2% glucose, and 5% glycerol. The RrF1β gene was amplified from R. rubrumgenomic DNA by PCR and cloned as described in (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar). Since the PCR is known to cause mutations, two PCR products, one obtained with Vent and the second with Taq DNA polymerase, were cloned and fully sequenced by the Taq dye deoxy chain termination method, using a 373A DNA Sequenase. Both clones were found to contain one mutated nucleotide. In the first clone, nucleotide 222 was changed, altering the published ACC codon of threonine 74 (18Falk G. Hampe A. Walker J.E. Biochem. J. 1985; 228: 391-407Crossref PubMed Scopus (94) Google Scholar) into an ACA one that also codes for threonine. So this clone encodes the WT RrF1β polypeptide. But the second clone had a mutation in nucleotide 584, which changed the GAG codon of glutamic acid 195 into the GGG codon of glycine. This clone thus encodes an RrF1β-E195G mutant polypeptide. Additional mutations at RrF1β-E195 were introduced by site-directed mutagenesis with the U.S.E. mutagenesis kit (Pharmacia Biotech Inc.) according to the instructions of the supplier, using theEcoRI-BamHI PCR-prepared fragment containing the complete WT RrF1β gene (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar) cloned into pUC18. The mutagenic primers, which must anneal to the same strand of the heat-denatured plasmid DNA as the U.S.E. selection primer, were therefore designed with the 5′-end to right. The oligonucleotides used for the E195K and E195Q substitutions were, respectively: 3′-A-GAA-ATA-GTG-TTC-TAC-TAG-CTA -CGG-CCC-TAA-T-5′ and 3′-A-GAA-ATA-GTG-GTC-TAC-TAG-CTA -CGG-CCC-TAA-T-5′. They contained a new, underlined site for ClaI, introduced by a single base change, indicated by a bold letter as the bases changed to give the Lys or Gln codons. The U.S.E. selection primer eliminates the ScaI site in the pUC18 amp gene, but the repair defective E. coliNM 522 mutS strain, transformed with theScaI-resistant mutated plasmid DNA according to the instructions of the supplier, failed to grow in liquid medium. The transformed cells did, however, form ampicillin-resistant colonies when plated on solid agar. Introduction of the mutations into the RrF1β gene was confirmed by hybridization of these colonies with a 32P-labeled mutagenic primer. The mutations were further confirmed by ClaI restriction analysis, using the ClaI site introduced into the mutagenic primers, and by full DNA sequencing. The β-E195Q gene had only the stated mutation. But β-E195K had two additional changes: 1) in nucleotide 159, altering the codon from GTG to GTT, both coding for valine 53 (18Falk G. Hampe A. Walker J.E. Biochem. J. 1985; 228: 391-407Crossref PubMed Scopus (94) Google Scholar); and 2) in nucleotide 1069, altering the codon from CTG to TTG, both coding for leucine 357 (18Falk G. Hampe A. Walker J.E. Biochem. J. 1985; 228: 391-407Crossref PubMed Scopus (94) Google Scholar). So this gene also encodes only the stated mutation. Cultures of the various transformed unc-depleted E. colistrains were centrifuged and resuspended in buffer containing 50 mm Tricine-NaOH, pH 8.0; 4 mm MgATP, and 10% glycerol, which is optimal for isolating active native RrF1β (25Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar). The protease inhibitors benzamidine, phenylmethanesulfonyl fluoride, andN α-p-tosyl-l-lysine chloromethyl ketone were added at 1.5 mm, 1 mm, and 30 μm, respectively, and the cells were disrupted under argon in a Yeda press (26Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar). The soluble cytoplasmic and insoluble membrane fractions were analyzed for the presence of expressed RrF1β by SDS-PAGE (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205325) Google Scholar, 32Flings S.P. Gregerson D.S. Anal. Biochem. 1986; 155: 83-88Crossref PubMed Scopus (772) Google Scholar) and Western immunoblotting (33Gershoni J.M. Methods Biochem. Anal. 1988; 33: 1-58PubMed Google Scholar). β-less chromatophores were obtained as described in (26Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar). This technique releases all their RrF1β (Refs. 25Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar and 34Philosoph S. Gromet-Elhanan Z. Eur. J. Biochem. 1981; 119: 107-113Crossref PubMed Scopus (14) Google Scholar; see Fig. 4, lane 6) together with trace amounts of RrF1α (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar, 35Gromet-Elhanan Z. Sokolov M. Photosynth. Res. 1995; 46: 79-86Crossref PubMed Scopus (6) Google Scholar), leading to loss of their ATP synthesis and hydrolysis activity. Reconstitution was carried out by incubating β-less chromatophores, at 5 μg of Bchl, for 1 h at 35 °C in 0.2 ml of a reaction mixture containing 50 mm Tricine-NaOH (pH 8.0), 25 mmMgCl2, 4 mm ATP, 1 mmdithiothreitol, and unless otherwise stated, 1 μg of RrF1α and saturating amounts of RrF1β at various stages of purification. ATP synthesis was usually assayed by a 5-fold dilution of the 0.2-ml mixture of reconstituted chromatophores into an assay mixture which contained in 1 ml a final concentration of 50 mmTricine-NaOH (pH 8.0), 5 mm MgCl2, 4 mm sodium phosphate (containing 0.4–0.8 × 106 cpm of 32Pi), 2 mmADP, 15 mm glucose, 24 units of hexokinase, and 66 μm N-methylphenazonium methosulfate. When assaying the Mg2+ requirement of the restored ATP synthesis (and hydrolysis), about 3 ml of the reconstituted chromatophores were subjected to two rounds of centrifugation and resuspension in 50 mm Tricine-NaOH (pH 8.0) and 10% glycerol to remove the 25 mm MgCl2, which are essential for the reconstitution (25Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar, 26Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar). These washed chromatophores, at 3–5 μg of Bchl, were added to the synthesis assay mixture, together with the indicated concentrations of MgCl2, and preequilibrated for 5 min at 35 °C in the dark. ATP synthesis was started by illumination and stopped after 3 min by turning off the lights and adding 0.1 ml of 2 m trichloroacetic acid. The synthesized [γ-32P]ATP was determined as described by Avron (36Avron M. Biochim. Biophys. Acta. 1960; 40: 257-272Crossref PubMed Scopus (500) Google Scholar). For assaying restored ATP hydrolysis, the washed chromatophores, at 3–5 μg of Bchl, were preincubated for 5 min at 35 °C in 0.66 ml of 50 mm Tricine-NaOH (pH 8.0) with the divalent cation concentrations indicated in the text. Hydrolysis was started by addition of 40 μl of ATP to a final concentration of 4 mmand stopped by 0.1 ml of 2 m trichloroacetic acid. The released Pi was measured according to (37Takeyama M. Ihara K. Moriyama Y. Ida K. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1953; 265: 21279-21284Abstract Full Text PDF Google Scholar). For measurement of ATP binding, purified RrF1β preparations were depleted of bound MgATP by elution-centrifugation through Sephadex G-50 columns (38Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar) preequilibrated with TGN buffer containing 50 mmTricine-NaOH (pH 8.0), 20% glycerol, and 50 mm NaCl. Binding was assayed by incubating 10 μm depleted RrF1β for 90 min at 23 °C in TG buffer with the indicated concentrations of MgCl2 and ATP, containing 1–2 × 106 cpm of [2,8-3H]ATP. Incubation was started by addition of RrF1β and stopped by subjecting 50-μl samples to elution-centrifugation, and the effluent was assayed for protein and radioactivity as described by Gromet-Elhanan and Kananshvili (23Gromet-Elhanan Z. Kananshvili D. Biochemistry. 1984; 23: 1022-1028Crossref Scopus (39) Google Scholar). Protein concentrations were measured either by the BCA method (39Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18305) Google Scholar) or according to Lowry et al. (40Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The Bchl content of chromatophores was determined at 880 nm using the absorption coefficient in vivo given by Clayton (41Clayton R.K. Gest H. San Pietro A. Vernon L.P. Bacterial Photosynthesis. Antioch Press, Yellow Springs, OH1963: 495-500Google Scholar). E. coli DK8 was a gift of Dr. M. Futai, Institute of Scientific and Industrial Research, Osaka University. E. coli LM2800 and LM3115 were a gift of Dr. P. R. Jensen, The Netherlands Cancer Institute, Amsterdam. Oligonucleotides were synthesized by Dr. Ora Goldberg, Biological Services, The Weizmann Institute of Science. Restriction enzymes, Vent DNA polymerase, and T4 ligase were from New England Biolabs.Taq DNA polymerase, and dNTP were purchased from Boehringer Mannheim. Plasmids pBSKS+ and pBTacI were from Stratagene and Boehringer Mannheim, respectively. The U.S.E. Mutagenesis Kit was obtained from Pharmacia Biotech Inc.. [2,8-3H]ATP was purchased from NEN Life Science Products. All other reagents were of the highest purity available. The β-E195G mutation was obtained as a mistake, which changed the GAG codon of glutamic acid to the GGG codon of glycine, during amplification of the RrF1β gene from genomic R. rubrum DNA by PCR. Since the equivalent MF1β-E199 is described in the crystal structure as pointing into the conical tunnel leading to the catalytic nucleotide binding site (8Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2707) Google Scholar), it was interesting to check whether the E195G mutation affects activity. Using the system described previously (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar), the expressed, partially purified mutated β subunit was found to restore a much lower rate of ATP synthesis in β-less chromatophores than the native or expressed WT RrF1β. We have therefore prepared two additional RrF1 β-E195 mutants by oligonucleotide-directed mutagenesis: from glutamic acid to lysine (β-E195K), carrying a positive charge, and to glutamine (β-E195Q), carrying no charge. Cloned WT and mutant β genes were ligated into the expression vector pBTacI, and the recombinant plasmid was transformed into E. coli LM3115 (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar). This strain was found to express larger amounts of RrF1β than two otherunc-deleted strains, LM2800 (29Jensen P.R. Michelsen O. J. Bacteriol. 1992; 174: 7635-7641Crossref PubMed Google Scholar) and DK8 (30Klionsky D.J. Brusilow W.S.A. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar), under all tested growth conditions (not shown). Optimal conditions for expression of large amounts of RrF1β as a soluble protein include growth of E. coli LM3115 at 22 °C in the presence of 5% glycerol to about A 0.65. Growth at higher temperatures or to a higher optical density increased the fraction of expressed β subunit appearing in inclusion bodies (42Nathanson, L. (1997) Cloning, Expression and Activity of Wild Type and Mutant B Subunits of the Rhodospirillum rubrum F0F1 ATP Synthase, Ph.D. thesis, The Weizmann Institute of Science.Google Scholar). The soluble cytoplasmic fraction of E. coli LM3115 cells containing the expressed WT and mutated RrF1β was subjected to ammonium sulfate fractionation as described for native RrF1β (34Philosoph S. Gromet-Elhanan Z. Eur. J. Biochem. 1981; 119: 107-113Crossref PubMed Scopus (14) Google Scholar). The 30–60% ammonium sulfate precipitate was dissolved in TGN buffer to a concentration of about 10 mg of protein/ml and thoroughly dialyzed against the same buffer. The WT and β-E195G mutant were further purified by dye-ligand chromatography on a Red A column. As is illustrated in Fig. 1, the dialyzed WT β subunit was loaded on a column preequilibrated in TGN buffer at 4 °C. After 1 h, the column was washed with TGN buffer, to elute all the unbound protein (Fig. 1, A,peak I, and B, lane 3). The RrF1β was then eluted with TGN buffer containing 1 mm MgATP (Fig. 1, A, peak II, andB, lane 4). Further washing with TGN buffer containing 1 mm MgATP and 1.5 m NaCl did not elute any additional RrF1β subunit (Fig. 1, A,peak III, and B, lane 5). This column yielded a highly purified expressed WT RrF1β (Fig. 1 B, lane 4), which contained, of course, no trace of RrF1α. On the other hand, the earlier prepared native RrF1β, which was removed by LiCl extraction ofR. rubrum chromatophores (25Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar) and purified by chromatography through two ion-exchange columns (26Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar), was recently found to contain 5–10% of RrF1α (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar, 35Gromet-Elhanan Z. Sokolov M. Photosynth. Res. 1995; 46: 79-86Crossref PubMed Scopus (6) Google Scholar). Most of this α subunit was removed by dye-ligand chromatography, leaving a pure native RrF1β containing less than 1% of RrF1α. All three types of RrF1β subunits were found to bind, in the presence of Mg2+, up to 2 mol of ATP/mol of β. But, whereas the binding of ATP to the expressed WT as to pure native RrF1β (Fig. 2), saturated at 200 μm ATP and showed a pronounced cooperativity, with a Hill coefficient (n H) of 2.2, the RrF1β-E195G mutant required a higher concentration of ATP for saturation and showed a much lower cooperativity with a Hill coefficient of only 1.5. The native RrF1β, which contained at least 5% of RrF1α (28Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar, 35Gromet-Elhanan Z. Sokolov M. Photosynth. Res. 1995; 46: 79-86Crossref PubMed Scopus (6) Google Scholar), was earlier shown to bind up to 2 mol of ATP/mol (23Gromet-Elhanan Z. Kananshvili D. Biochemistry. 1984; 23: 1022-1028Crossref Scopus (39) Go
Referência(s)