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• W2016736231 abstract "In F1F0-ATP synthase, the subunit b2δ complex comprises the peripheral stator bound to subunit a in F0 and to the α3β3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at γCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9–10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly. In F1F0-ATP synthase, the subunit b2δ complex comprises the peripheral stator bound to subunit a in F0 and to the α3β3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at γCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9–10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly. F-type ATPases (F1F0) are ubiquitously abundant in the inner membranes of mitochondria, chloroplasts, and bacteria, where they catalyze the synthesis of ATP by oxidative or photophosphorylation. In bacteria, the enzyme can also work in the opposite direction to generate proton or sodium gradients at the expense of ATP. Despite slight variations in subunit composition among species, F1F0 complexes share a high homology with respect to the mechanism of catalysis, in which ion translocation through the membrane-embedded F0 part is rotationally coupled to ATP synthesis/hydrolysis in F1 (1Weber J. Senior A.E. FEBS Lett. 2003; 545: 61-70Crossref PubMed Scopus (239) Google Scholar). Because of the rotary mechanics, in addition to the structural classification of this multisubunit enzyme complex in F1 (subunit composition α3β3γδϵ in Escherichia coli) and F0 (ab2c10) (2Fillingame R.H. Dmitriev O.Y. Biochim. Biophys. Acta. 2002; 1565: 232-245Crossref PubMed Scopus (65) Google Scholar), a functional classification into rotor and stator is also used. Either H+translocation through F0 or ATP hydrolysis in F1 leads to the rotary movement of a centrally located γϵc10 rotor element (3Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar, 4Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar, 5Junge W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4735-4737Crossref PubMed Scopus (90) Google Scholar, 6Yasuda R. Noji H. Yoshida M. Kinosita Jr., K. Itoh H. Nature. 2001; 410: 898-904Crossref PubMed Scopus (711) Google Scholar, 7Diez M. Zimmermann B. Börsch M. König M. Schweinberger E. Steigmiller S. Reuter R. Felekyan S. Kudryavtsev V. Seidel C.A. Gräber P. Nat. Struct. Mol. Biol. 2004; 11: 135-141Crossref PubMed Scopus (344) Google Scholar, 8Zimmermann B. Diez M. Nawid Z. Gräber P. Börsch M. EMBO J. 2005; 24: 2053-2063Crossref PubMed Scopus (98) Google Scholar), which has to be counteracted by a peripheral stator element. This so-called “second stalk” is composed at least of the two copies of subunit b (9Wilkens S. Capaldi R.A. Nature. 1998; 393: 29Crossref PubMed Scopus (135) Google Scholar, 10McLachlin D.T. Coveny A.M. Clark S.M. Dunn S.D. J. Biol. Chem. 2000; 275: 17571-17577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), which are supposed to undergo transient elastic deformation to compensate for the torque, which is built up by the propelling rotor (5Junge W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4735-4737Crossref PubMed Scopus (90) Google Scholar, 11Greie J.-C. Deckers-Hebestreit G. Altendorf K. Eur. J. Biochem. 2000; 267: 3040-3048Crossref PubMed Scopus (21) Google Scholar, 12Altendorf K. Stalz W.-D. Greie J.-C. Deckers-Hebestreit G. J. Exp. Biol. 2000; 203: 19-28PubMed Google Scholar). Accordingly, a similar mode of elastic coupling during catalysis has recently been suggested for the subunit c ring of the rotor part from the Na+-translocating ATP synthase of Ilyobacter tartaricus (13Meier T. Polzer P. Diederichs K. Welte W. Dimroth P. Science. 2005; 308: 659-662Crossref PubMed Scopus (325) Google Scholar). The peripheral connection between F1 and F0 by subunit b is accomplished by multiple contacts of the subunit b dimer with the α, β, and δ subunits of F1 (10McLachlin D.T. Coveny A.M. Clark S.M. Dunn S.D. J. Biol. Chem. 2000; 275: 17571-17577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 14Dunn S.D. Revington M. Cipriano D.J. Shilton B.H. J. Bioenerg. Biomembr. 2000; 32: 347-355Crossref PubMed Scopus (41) Google Scholar, 15McLachlin D.T. Dunn S.D. Biochemistry. 2000; 39: 3486-3490Crossref PubMed Scopus (28) Google Scholar) as well as with subunit a of F0 (16Fillingame R.H. Jiang W. Dmitriev O.Y. Jones P.C. Biochim. Biophys. Acta. 2000; 1458: 387-403Crossref PubMed Scopus (56) Google Scholar, 17Greie J.-C. Deckers-Hebestreit G. Altendorf K. J. Bioenerg. Biomembr. 2000; 32: 357-364Crossref PubMed Scopus (18) Google Scholar, 18Long J.C. DeLeon-Rangel J. Vik S.B. J. Biol. Chem. 2002; 277: 27288-27293Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Because of the transient storage of elastic energy during catalysis, subunit interactions between components of the stator have to be rather strong to withstand a rotary strain of up to 55 kJ mol-1, i.e. the maximum ΔG observed for ATP synthesis (19Pänke O. Cherepanov D.A. Gumbiowski K. Engelbrecht S. Junge W. Biophys. J. 2001; 81: 1220-1233Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 20Diez M. Börsch M. Zimmermann B. Turina P. Dunn S.D. Gräber P. Biochemistry. 2004; 43: 1054-1064Crossref PubMed Scopus (40) Google Scholar). Although there are several binding partners for subunit b within the stator in F1, each of which contributes to binding affinity (21Dunn S.D. Chandler J. J. Biol. Chem. 1998; 273: 8646-8651Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 22Weber J. Wilke-Mounts S. Nadanaciva S. Senior A.E. J. Biol. Chem. 2004; 279: 11253-11258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), in the case of F0, only subunit a interacts with the subunit b dimer. Although binding affinities between subunits a and b could so far not be determined within the lipid phase, a strong interaction has been shown by the purification of a stable ab2 subcomplex (23Stalz W.-D. Greie J.-C. Deckers-Hebestreit G. Altendorf K. J. Biol. Chem. 2003; 278: 27068-27071Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In the case of the interaction of subunit b with F1, binding affinities have so far been determined only in solution by several techniques, including fluorometric tryptophan quenching (1Weber J. Senior A.E. FEBS Lett. 2003; 545: 61-70Crossref PubMed Scopus (239) Google Scholar, 22Weber J. Wilke-Mounts S. Nadanaciva S. Senior A.E. J. Biol. Chem. 2004; 279: 11253-11258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 24Weber J. Muharemagic A. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2003; 278: 13623-13626Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and fluorescence resonance energy transfer (FRET) 6The abbreviations used are: FRET, fluorescence resonance energy transfer; DDM, n-dodecyl β-d-maltoside; TMR, tetramethylrhodamine-5-maleimide; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FCS, fluorescence correlation spectroscopy. (20Diez M. Börsch M. Zimmermann B. Turina P. Dunn S.D. Gräber P. Biochemistry. 2004; 43: 1054-1064Crossref PubMed Scopus (40) Google Scholar). However, in these assays, only truncated forms of subunit b lacking the membrane part were used, thereby confusing the interpretation of the corresponding results with a rather weak dissociation constant for dimerization (20Diez M. Börsch M. Zimmermann B. Turina P. Dunn S.D. Gräber P. Biochemistry. 2004; 43: 1054-1064Crossref PubMed Scopus (40) Google Scholar, 22Weber J. Wilke-Mounts S. Nadanaciva S. Senior A.E. J. Biol. Chem. 2004; 279: 11253-11258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Subunit b dimerization was shown to be a prerequisite for F1 binding (25Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar), and the two copies of subunit b were shown to interact also within the transmembrane portion of the polypeptide (26Dmitriev O. Jones P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1999; 274: 15598-15604Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In addition, the use of soluble F1 and single F0 subunits in titration assays does not allow testing of functional F1/F0 interactions because of the lack of the membrane-embedded F0 part of the enzyme. It has previously been shown that, in the case of reconstituted F0 and its subcomplexes, all three subunits a, b, and c are necessary for the functional binding of F1 (11Greie J.-C. Deckers-Hebestreit G. Altendorf K. Eur. J. Biochem. 2000; 267: 3040-3048Crossref PubMed Scopus (21) Google Scholar, 23Stalz W.-D. Greie J.-C. Deckers-Hebestreit G. Altendorf K. J. Biol. Chem. 2003; 278: 27068-27071Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 27Greie J.-C. Heitkamp T. Altendorf K. Eur. J. Biochem. 2004; 271: 3036-3042Crossref PubMed Scopus (22) Google Scholar). Thus, both rotor (subunit c) and stator (subunit b) components of F0 contribute to F1 binding in vivo. In this study, F1/F0 interactions were quantified for the first time using functionally reconstituted protein complexes. The binding of F1 to the subunit b dimer and ab2 stator subcomplexes as well as to fully assembled F0 has been observed by single-molecule FRET, also introducing a new approach in the spectroscopic analysis of binding constants in F1/F0 interaction. The binding constants clearly demonstrate that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. Construction of Plasmids and Growth Conditions—Plasmid pTOM3.1 was constructed by cloning a 144-bp EcoNI fragment from pSK1 (28Kauffer S. Deckers-Hebestreit G. Altendorf K. Eur. J. Biochem. 1991; 202: 1307-1312Crossref PubMed Scopus (4) Google Scholar) as well as a 478-bp PpuMI/BssHI fragment from pRR76 (29Börsch M. Diez M. Zimmermann B. Reuter R. Gräber P. FEBS Lett. 2002; 527: 147-152Crossref PubMed Scopus (114) Google Scholar) into plasmid pBWU13 (atpI′BEFHAGDC) (30Iwamoto A. Omote H. Hanada H. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 16350-16355Abstract Full Text PDF PubMed Google Scholar), thereby introducing the substitutions bC21A and bQ64C. Addition of a polyhistidine motif following the N-terminal methionine residue of subunit a was achieved by the site-directed introduction of a (CATCAC)6 sequence via a two-stage PCR mutagenesis procedure (31Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar), yielding plasmid pTOM3.1aHis12. Both plasmids were transformed into E. coli strain DK8 (ΔatpBEFHAGDC) (32Klionsky D.J. Brusilow W.S. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar), and cultures were grown on minimal medium with glycerol as the carbon source (11Greie J.-C. Deckers-Hebestreit G. Altendorf K. Eur. J. Biochem. 2000; 267: 3040-3048Crossref PubMed Scopus (21) Google Scholar). Cells were harvested at late exponential phase and stored at -80 °C. Preparative Methods—The preparation of F1 from E. coli RA1/pRA114 (33Gogol E.P. Lücken U. Bork T. Capaldi R.A. Biochemistry. 1989; 28: 4709-4716Crossref PubMed Scopus (72) Google Scholar, 34Aggeler R. Capaldi R.A. J. Biol. Chem. 1992; 267: 21355-21359Abstract Full Text PDF PubMed Google Scholar) containing the mutation γT106C was carried out as described (33Gogol E.P. Lücken U. Bork T. Capaldi R.A. Biochemistry. 1989; 28: 4709-4716Crossref PubMed Scopus (72) Google Scholar). F0 and subunit b from DK8/pTOM3.1 were isolated as described previously (11Greie J.-C. Deckers-Hebestreit G. Altendorf K. Eur. J. Biochem. 2000; 267: 3040-3048Crossref PubMed Scopus (21) Google Scholar, 35Schneider E. Altendorf K. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7279-7283Crossref PubMed Scopus (50) Google Scholar). To purify the ab2 subcomplex, everted membrane vesicles were prepared at 4 °C by resuspending 50 g of DK8/pTOM3.1aHis12 cells in 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, and 10 μg/ml DNase, followed by cell disruption with a Constant Systems Basic Z cell disrupter (IUL Instruments GmbH) at a pressure of 1.36 kilobars. The membrane suspension was centrifuged at 15,000 × g for 30 min. To separate everted membrane vesicles from the cytosolic fraction, the supernatant was centrifuged at 150,000 × g for 1.5 h. To remove F1, membranes were washed with 10 mm Tris-HCl (pH 7.5), 10 mm EDTA, and 10% (v/v) glycerol; resuspended in 1 mm Tris-HCl (pH 7.5), 6 m urea, and 10% (v/v) glycerol; and incubated overnight. Membranes were collected by centrifugation and washed with 50 mm Tris-HCl (pH 7.5) and 10% (v/v) glycerol. For solubilization, membranes (10 mg/ml) were stirred with 1.4% (w/v) n-dodecyl β-d-maltoside (DDM) (Glycon Corp.) at 4 °C for 1 h and subsequently centrifuged at 232,000 × g for 15 min. The supernatant was adjusted to 150 mm NaCl, 10 mm imidazole, and 0.1 mm phenylmethylsulfonyl fluoride and incubated with 1 ml of nickel-nitrilotriacetic acid-agarose (Qiagen Inc.)/10 mg of membrane protein at 4 °C for 1 h. The agarose matrix was pre-equilibrated with 50 mm Tris-HCl (pH 7.5), 10% (v/v) glycerol, 150 mm NaCl, 10 mm imidazole, 0.05% (w/v) DDM, and 0.1 mm phenylmethylsulfonyl fluoride. The agarose was then packed into an empty glass column and washed with 5–10 column volumes of the equilibration buffer. To remove unspecifically bound protein, the imidazole concentration was temporarily increased to 60 mm for 5–10 column volumes, followed by a decrease to 10 mm for another 5–10 column volumes. Detergent was exchanged from DDM to Na+ cholate using 5–10 column volumes of the equilibration buffer containing 1% (w/v) Na+ cholate instead of DDM. Elution of the ab2 subcomplex with 250 mm imidazole was preceded by a gradient from 10 to 55 mm imidazole within 10 column volumes. Eluted protein was concentrated to 0.5–1 mg/ml using Amicon Ultra-4 centrifugal filter devices (molecular weight cutoff of 10,000; Millipore Corp.) and dialyzed against a 1000-fold volume of 50 mm Tris-HCl (pH 7.5), 10% (v/v) glycerol, 150 mm NaCl, 10 mm imidazole, 1% (w/v) Na+ cholate, and 0.1 mm phenylmethylsulfonyl fluoride for 24 h with changing the buffer once. Labeling F0 Components and F1—Isolated F0, subunit b, and ab2 subcomplex were labeled with tetramethylrhodamine-5-maleimide (TMR) (Molecular Probes, Inc.), whereas purified F1 was labeled with Cy5-maleimide (referred to as Cy5; Amersham Biosciences). The dyes were determined after a 1000-fold dilution with methanol using the extinctioncoefficients provided by the supplier. The Forster radius (R0) for this FRET pair is ∼6.4 nm (7Diez M. Zimmermann B. Börsch M. König M. Schweinberger E. Steigmiller S. Reuter R. Felekyan S. Kudryavtsev V. Seidel C.A. Gräber P. Nat. Struct. Mol. Biol. 2004; 11: 135-141Crossref PubMed Scopus (344) Google Scholar). F0 and subcomplexes thereof were labeled at bCys64 with TMR in 10 mm Tris/NaOH (pH 8.0), 150 mm NaCl, 1% (w/v) Na+cholate, and 10% (v/v) glycerol on ice in the presence of a 5-fold excess of tris(2-carboxyethyl)phosphine hydrochloride (Molecular Probes, Inc.) with respect to protein to prevent the formation of disulfides. To avoid the labeling of both b subunits within the dimer, the degree of labeling was adjusted to ∼35% by applying the fluorescent dye at different molar ratios and incubation times, i.e. for F0, a molar ratio of 1:1 for 3 h; for the ab2 subcomplex, a molar ratio of 1:5 for 4.5 h; and for subunit b, a molar ratio of 1:5 for 1.5 h. In the latter case, 50 mm MOPS (pH 7.0), 100 mm NaCl, 100 μm MgCl2, and 0.1% (w/v) DDM was used. Unbound dye and tris(2-carboxyethyl)phosphine hydrochloride were removed using pre-equilibrated Sephadex G-50 columns (Amersham Biosciences). The labeling degrees (β) were calculated from the concentration ratio of bound dye and protein according to the following: β = ([labeled protein]/[total protein]) × 100% = ((A556/ϵ556(TMR))/(A278(protein)/ϵ278(protein))) × 100% and A278(protein) = A278(total) - A278(TMR) = (A278(total) - (ϵ278(TMR)/ϵ556(TMR)) × A556, where A278 and A556 are the absorbance at 278 and 556 nm, respectively; and ϵ278 and ϵ556 are the extinction coefficients at 278 and 556 nm, respectively. The concentrations were determined from UV-visible absorption spectra of the labeled proteins using ϵ278(F0) = 136,000 m-1 cm-1, ϵ278(ab2) = = 108,000 m cm-1, ϵ278(TMR) = 19,500 m-1 cm-1, and ϵ556(TMR) = 95,000 M-1 cm-1, yielding TMR labeling rates of 32% for F0 and 36% for the ab2 subcomplex. The rather low absorbance of subunit b (ϵ278 = 7100 m-1 cm-1) was masked by the absorbance of TMR itself. Thus, for TMR-labeled subunit b, the protein concentration was determined with the enhanced BCA protein assay (Pierce) using unlabeled subunit b as a standard. The TMR concentration was then measured by UV-visible spectroscopic absorption analysis, from which a labeling degree of 29% was calculated. F1 was labeled at γCys106 with Cy5 at a molar ratio of 1:0.9 in 50 mm MOPS/NaOH (pH 7.0) and 100 μm MgCl2 on ice for 4 min (36Börsch M. Turina P. Eggeling C. Fries J.R. Seidel C.A. Labahn A. Gräber P. FEBS Lett. 1998; 437: 251-254Crossref PubMed Scopus (78) Google Scholar). Unbound dye was removed by gel filtration on Sephadex G-50. A labeling degree (α) of ∼58% was calculated from UV-visible absorption spectra (for details, see above) using ϵ650(Cy5) = 250,000 m-1 cm-1, ϵ278(Cy5) = 41,100 m-1 cm-1, and ϵ278(F1) = 205,500 m-1 cm-1. Solutions of labeled protein were frozen in liquid nitrogen after addition of 10% (v/v) glycerol and stored at -80 °C. Reconstitution of F0, the ab2 Subcomplex, and Subunit b into Liposomes and Reassembly with F1—Liposomes from phosphatidylcholine and phosphatidic acid were prepared by dialysis (37Fischer S. Gräber P. FEBS Lett. 1999; 457: 327-332Crossref PubMed Scopus (83) Google Scholar). TMR-labeled F0, ab2 subcomplex, and subunit b were reconstituted according to Fischer et al. (38Fischer S. Etzold C. Turina P. Deckers-Hebestreit G. Altendorf K. Gräber P. Eur. J. Biochem. 1994; 225: 167-172Crossref PubMed Scopus (64) Google Scholar). The final concentration of proteoliposomes was 8 mg/ml of lipid in 20 mm Tricine/NaOH (pH 8.0), 20 mm succinate, 2.5 mm MgCl2, and 0.6 mm KOH. For the determination of catalytic activities and ensemble fluorescence measurements, the enzyme concentration was adjusted to 40 nm. In the case of single-molecule fluorescence measurements, the concentration of reconstituted protein was 15 nm, resulting in an average number of less than one enzyme molecule/liposome (8Zimmermann B. Diez M. Nawid Z. Gräber P. Börsch M. EMBO J. 2005; 24: 2053-2063Crossref PubMed Scopus (98) Google Scholar). Proteoliposomes were incubated with different concentrations of labeled F1 (0, 0.09, 0.9, 9, 49, 89, 222, and 444 nm) in the presence of 2.5 mm MgCl2 and 50 mm NaCl for 45 min at 37 °C, followed by a 90-min incubation on ice. Unbound F1 was removed by subsequent centrifugation at 265,000 × g for 90 min, and the pellet was resuspended in 20 mm Tricine/NaOH (pH 8.0), 20 mm succinate, 0.6 mm KCl, 2.5 mm MgCl2, and 4% (v/v) glycerol. Fluorescence Measurements—Ensemble fluorescence measurements were performed at 20 °C using an SLM-AMINCO 8100 spectrofluorometer with a slit width of 4 nm. Spectra were corrected for lamp intensity and detection efficiency. Single-molecule fluorescence measurements were performed at 20 °C using a confocal microscope (100-μm pinhole size) of local design. The laser beam (532 nm, frequency-doubled neodymium/yttrium aluminum garnet; Coherent Inc.) was attenuated to 100 microwatts and directed into an Olympus water immersion objective (UApo 40×, numerical aperture of 1.15). This power level created sufficiently high fluorescence signals, but still kept photobleaching negligible. For epiillumination, a 545 nm DCLP dichroic mirror (AHF Corp.) was used. Fluorescence was subdivided by a 630 nm DCLP dichroic mirror into two spectral ranges with λ < 630 nm for TMR and λ > 630 nm for Cy5 and detected with two avalanche photodiodes (SPCM-AQR 151, EG&G). Filters (HQ 575/65 nm for TMR and HQ 665 nm LP for Cy5) were used to block laser light scattering and to reduce the cross-talk of TMR into the Cy5 detection channel to 5.4%. The excitation efficiency of Cy5 at 532 nm was <0.03 times that of TMR. Photons were recorded simultaneously (1-ms time resolution) with a multiscaler photon counter (PMS-300, Becker & Hickl GmbH). Samples were analyzed on a microscope slide with a cavity of ∼85 μl covered with a cover glass. Labeled proteoliposomes were diluted to a final concentration of ∼100 pm in 20 mm Tricine/NaOH (pH 8.0), 20 mm succinate, 0.6 mm KCl, and 2.5 mm MgCl2. At this concentration, one liposome at most was present in the confocal volume at the same time. For fluorescence correlation spectroscopy (FCS), only the photons of the TMR channel were used to calculate the autocorrelation function (G(τc)) by an ALV 5000/E FAST real-time correlator. For a quantitative interpretation, we used the following function, which contains a diffusion term and a contribution of one triplet state (Equation 1),G(τc)=1+1NF11+τc/τD·11+(ω0/Z0)2τc/τD1/2·(1-T+T(-τc/τT))(Eq. 1) where G(τc) is the autocorrelation function; NF is the average number of fluorescent molecules in the detection volume; τc is the correlation time; τD = ω20/4D is the characteristic time of diffusion with D (diffusion coefficient); τT is the characteristic triplet time; T is the average fraction of molecules in the excited triplet state; and ω0 and z0 are the 1/e2 radii of the gaussian detection volume in the radial and axial directions, respectively. The actual confocal detection volume (V = 7.7 fl) was calculated from the FCS data of rhodamine 6G in water as described (36Börsch M. Turina P. Eggeling C. Fries J.R. Seidel C.A. Labahn A. Gräber P. FEBS Lett. 1998; 437: 251-254Crossref PubMed Scopus (78) Google Scholar). For FCS, samples were diluted to a final concentration of 2–5 nm, which yielded a mean value of 5–10 molecules within the confocal volume at the same time. For every single-molecule FRET titration experiment, three independent measurements were performed. To determine the diffusion times (τD), the autocorrelation functions were fitted by Equation 1. All best fits of FCS data resulted in similar values for the triplet contribution, i.e. τT (4 μs) and T (0.03–0.07). Determination of Kd by Single-molecule FRET Analysis—Single-molecule FRET data were analyzed by the custom-made software Burst Analyzer. After correction of background count rates (0.5–2 counts/ms) and cross-talk of TMR into the Cy5 channel, photon bursts were selected by the following criteria. 1) A duration time of >20 ms identified photon bursts originating from labeled proteoliposomes with a corresponding mean diffusion time through the confocal detection volume. 2) Count rates higher than 10 photons/ms for the TMR channel or higher than seven photons/ms for the Cy5 channel enabled the unambiguous determination of the presence of both fluorophores in the proteoliposome. 3) Photon bursts were excluded from further analysis if the total count rate, i.e. the sum of photons in the donor and acceptor channels, was >7000 because these bursts presumably indicate aggregates of liposomes. For each selected burst, the apparent mean FRET efficiency was calculated by Eapp = IA/(IA + ID), with IA and ID being the corrected intensities of Cy5 (acceptor) and TMR (donor), respectively. Bursts were classified as either donor-only events (Eapp ≤ 0.05) or FRET events (Eapp > 0.05), and the ratio of FRET events to all events was calculated and plotted against the F1 concentration. Assays—Protein concentrations were determined either by the BCA assay used as recommended by the supplier or by UV absorption spectroscopy using the extinction coefficients given above (39Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5060) Google Scholar). Proteins were separated by SDS-PAGE (16.5% T and 6% C separating gels together with 4% T and 3% C stacking gels) (40Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar) and detected by silver staining (41Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3741) Google Scholar). Specificity of subunit labeling was controlled by fluorescence detection of protein bands. ATPase activities were measured in an ATP-regenerating system (42Fischer S. Gräber P. Turina P. J. Biol. Chem. 2000; 275: 30157-30162Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) at 37 °C in 100 mm Tris-HCl (pH 8.0), 25 mm KCl, 4 mm MgCl2, 2.5 mm phosphoenolpyruvate, 18 units/ml pyruvate kinase, 16 units/ml lactate dehydrogenase, and 0.2 mm NADH. ATP synthesis was measured after an acid-base transition in the presence of an additional K+/valinomycin diffusion potential at room temperature (43Turina P. Samoray D. Gräber P. EMBO J. 2003; 22: 418-426Crossref PubMed Scopus (129) Google Scholar). 20 μl of F1F0 liposomes (40 nm) were incubated for 3 min with 80 μl of 20 mm succinate/NaOH (pH 4.7), 5 mm NaH2PO4, 0.6 mm KOH, 2.5 mm MgCl2, 100 μm ADP, and 20 μm valinomycin. 100 μl of the acidified suspension were then mixed with 900 μl of 200 mm Tricine/NaOH (pH 8.8), 5 mm NaH2PO4, 160 mm KOH, 2.5 mm MgCl2, and 100 μm ADP. The formation of ATP was monitored with a luciferin/luciferase assay. Purification and Fluorescence Labeling of Proteins—To observe binding of F1 to F0 and components thereof by intramolecular FRET, it was necessary to specifically label one subunit of each of the binding partners. All subunits were isolated and labeled with TMR (F0, ab2 subcomplex, and subunit b) or with Cy5 (F1) as described under “Experimental Procedures” (Fig. 1). Labeling degrees were determined by UV-visible absorption spectroscopy as described under “Experimental Procedures” and calculated to be 29–36% for TMR-labeled bCys64 in F0, the ab2 subcomplex, and subunit b and 58% for Cy5-labeled F1 γCys106. For the ab2 subcomplex and subunit b, UV illumination of the SDS gel revealed an additional slightly fluorescent protein band corresponding to the subunit b dimer. In the case of F0, the remaining impurities did not superimpose on the fluorescence of TMR at bCys64, which was required for the FRET analysis. γCys106 of purified F1 showed highly specific labeling with Cy5. The silver-stained SDS gel also revealed partial degradation of subunit δ, which has already been observed as a common problem in F1 preparations (44Rodgers A.J. Wilkens S. Aggeler R. Morris M.B. Howitt S.M. Capaldi R.A. J. Biol. Chem. 1997; 272: 31058-31064Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Functionality of Labeled Proteins—Previous studies revealed the functionality of isolated F0, ab2 subcomplex, and subunit b by passive proton translocation through F0 reconstituted from subcomplexes as well as from single subunits (11Greie J.-C. Deckers-Hebestreit G. Altendorf K. Eur. J. Biochem. 2000; 267: 3040-3048Crossref PubMed Scopus (21) Google Scholar, 23Stalz W.-D. Greie J.-C. Deckers-Hebestreit G. Altendorf K. J. Biol. Chem. 2003; 278: 27068-27071Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 27Greie J.-C. Heitkamp T. Altendorf K. Eur. J. Biochem. 2004; 271: 3036-3042Crossref PubMed Scopus (22) Google Scholar). To exclude a possible influence of the dye on the catalytic function of F1 and the coupling to F0, the rates of ATP synthesis and hydrolysis were determined with both the labeled and unlabeled enzymes. Isolated F0 was reconstituted into liposomes; and after binding of F1, all samples revealed nearly the same rate of ATP synthesis of ∼15 s-1 (TABLE ONE). Accordingly, ATP hydrolysis turnover rates of ∼120 s-1 were determined for isolated F1, whether Cy5-labeled or not. Both assays clearly demonstrate that the functionality of F1 and F0 was not affected by the" @default.
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• W2016736231 title "Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase" @default.
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• W2016736231 doi "https://doi.org/10.1074/jbc.m506251200" @default.
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