An intermediate step in the evolution of ATPases – a hybrid F 0 –V 0 rotor in a bacterial Na + F 1 F 0 ATP synthase Michael Fritz 1, *, Adriana L. Klyszejko 2, *, Nina Morgner 3, *, Janet Vonck 4 , Bernd Brutschy 3 , Daniel J. Muller 2 , Thomas Meier 4 and Volker Mu ¨ ller 1 1 Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt ⁄ Main, Germany 2 BioTechnological Center, University of Technology Dresden, Germany 3 Microkinetic, Clusterchemistry, Mass- and Laserspectroscopy, Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe University Frankfurt ⁄ Main, Germany 4 Max-Planck-Institute of Biophysics, Frankfurt, Germany ATP synthases are key elements in bioenergetics [1]. In bacteria, ATP synthesis is catalyzed by F 1 F 0 ATP syn- thase, which uses the electrochemical H + (or in some species Na + ) potential to drive the synthesis of ATP [2]. ATP synthases are rotary machines that work as a pair of coupled motors, a chemically driven motor (F 1 ) and a membrane-embedded, ion gradient-driven motor (F 0 ) [3]. The membrane-embedded motor comprises a stator and a rotor. The stator is formed by subunits a and b 2 , and the rotor is formed from multiple copies of subunit c. They form an oligomeric ring of non- covalently linked subunits, and rotation of the c ring is obligatorily coupled to ion flow across the membrane [4–6]. Subunit c of the F 1 F 0 ATP synthases has a molecular mass of approximately 8 kDa, and folds in the mem- brane like a hairpin, with two transmembrane helices connected by a cytoplasmic loop [7]. Each monomer contains an ion-binding site (H + or Na + ) [8,9]. Recent studies have demonstrated that the c ring stoichiometry in different organisms ranges between 10 and 15 mono- mers (see Discussion). Assuming that each subunit takes up one ion, each c ring revolution induces the synthesis of three molecules of ATP. This gives a Keywords Acetobacterium; acetogen; ATP-synthase; c ring; F 0 -V 0 hybrid rotor Correspondence V. Mu ¨ ller, Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Max-von-Laue-Straße 9, D-60438 Frankfurt, Germany Fax: +49 69 79829306 Tel: +49 69 79829507 E-mail: vmueller@bio.uni-frankfurt.de *These authors contributed equally to this study (Received 18 December 2007, revised 15 February 2008, accepted 22 February 2008) doi:10.1111/j.1742-4658.2008.06354.x The Na + F 1 F 0 ATP synthase operon of the anaerobic, acetogenic bacte- rium Acetobacterium woodii is unique because it encodes two types of c subunits, two identical 8 kDa bacterial F 0 -like c subunits (c 2 and c 3 ), with two transmembrane helices, and a 18 kDa eukaryal V 0 -like (c 1 ) c subunit, with four transmembrane helices but only one binding site. To determine whether both types of rotor subunits are present in the same c ring, we have isolated and studied the composition of the c ring. High- resolution atomic force microscopy of 2D crystals revealed 11 domains, each corresponding to two transmembrane helices. A projection map derived from electron micrographs, calculated to 5 A ˚ resolution, revealed that each c ring contains two concentric, slightly staggered, packed rings, each composed of 11 densities, representing 22 transmembrane helices. The inner and outer diameters of the rings, measured at the density bor- ders, are approximately 17 and 50 A ˚ . Mass determination by laser- induced liquid beam ion desorption provided evidence that the c rings contain both types of c subunits. The stoichiometry for c 2 ⁄ c 3 : c 1 was 9 : 1. Furthermore, this stoichiometry was independent of the carbon source of the growth medium. These analyses clearly demonstrate, for the first time, an F 0 –V 0 hybrid motor in an ATP synthase. Abbreviations AFM, atomic force spectroscopy; LILBID-MS, laser-induced liquid beam ion desorption mass spectroscopy. FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 1999 theoretical H + (Na + ) ⁄ ATP ratio of 3.5–5, which is the value required for ATP synthesis given a transmem- brane electrochemical ion gradient (Dl H + ⁄ Na + )of around )200 mV and a phosphorylation potential (DG p )of60kJÆmol )1 according to the equation: DG p ¼ nFDl Na þ where n is the number of translocated ions and F is the Faraday constant. The c subunit of the eukaryal V 1 V 0 ATPases present in organelles arose by duplication and fusion of the bacterial c subunit, giving rise to a protein of approxi- mately 16 kDa with four transmembrane helices that form two covalently linked hairpins in the membrane [10]. Importantly, the ion-binding site is not conserved in hairpin one. If one assumes the same number of transmembrane helices in V 0 and F 0 , the rotor of eukaryal V 1 V 0 ATPases has only half the number of ion-binding sites compared to F 1 F 0 ATP syntheses. This low H + (Na + ) ⁄ ATP ratio is apparently the reason for the inability of eukaryal V 1 V 0 ATPases to catalyze ATP synthesis in vivo [11,12]. On the other hand, this low ratio strongly favors the generation of steep ion gradients driven by ATP hydrolysis, and indeed the cellular function of V 1 V 0 ATPases is to energize endo- cytoplasmatic membranes in the eukaryotic cell [13]. The Na + F 1 F 0 ATP synthase operon from the anaerobic, acetogenic bacterium Acetobacterium woodii differs from all other F 1 F 0 ATP synthases by the presence of one V 0 subunit c gene (atpE 1 ) and two genes (atpE 2 ⁄ atpE 3 ) encoding identical F 0 c subunits. The gene atpE 1 encodes an 18 kDa protein with two predicted hairpins, and, like its eukaryotic counter- part, is missing one ion binding site (in hairpin two). The genes atpE 2 and atpE 3 encode two identical 8 kDa subunits with one ion-binding site each. The three genes are encoded in the same operon and their products are present in the same enzyme preparation [14–16]. Here, we have addressed the question whether both types of c subunit assemble into one ring, and whether the c ring composition changes with the growth conditions. We present data that unequivocally demonstrate a V 0 –F 0 hybrid rotor, the first found in nature. Results Purification of the c ring from A. woodii The c rings of the F 1 F 0 ATP synthase from A. woodii were isolated according to the method developed for Ilyobacter tartaricus [17]. The purified c ring migrated as a single band on SDS–PAGE, with an apparent molecular mass of 57–59 kDa, depending on the acryl- amide concentration used (Fig. 1A). Western blotting analyses of the intact as well as the denatured c rings revealed the presence of subunit c 1 as well as c 2 ⁄ 3 in the isolated c ring. Densitometric analysis of the c monomers visualized by silver staining (Fig. 1) revealed a stoichiometry of approximately 1 : 8.6 for c 1 : c 2 ⁄ 3 . As observed before for the c rings of entire Na + F 1 F 0 ATP synthases [18,19] as well as isolated c rings [20], the A. woodii c ring was highly stable and did not dissociate by boiling in 20 mm Tris ⁄ HCl, 5% SDS (pH 8.0) for up to 30 min, but did dissociate by autoclaving (120 °C) in the presence of 5% SDS for 5 min or in the presence of 40 mm trichloroacetic acid. The c rings from different preparations always showed the same migration behaviour in SDS–PAGE, and the isolated c ring migrated to a position identical to that of the c ring present in native ATP synthase solubi- lized in the same detergent (Fig. 1). Fig. 1. Isolation and subunit composition of the c rings from the Na + F 1 F 0 ATP synthase from A. woodii. Samples of isolated enzyme (lane 1) and isolated c rings (lanes 3, 4 and 7) were boiled at 80 °C for 20 min and applied to a 10.0% (lanes 1–3) or 13.5% (lanes 4–9) polyacrylamide gel. The c ring was disintegrated by treatment with trichloroacetic acid (lanes 6, 8 and 9), and individual subunits were detected by silver staining (lane 6) or immunoblotting using an antibody against subunit c 1 (lanes 7 and 8) or subunit c 2 ⁄ 3 (lane 9). The antibody against c 2 ⁄ 3 also reacts with c 1 . Hybrid V 0 –F 0 rotor in a F 1 F 0 ATP synthase M. Fritz et al. 2000 FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS High-resolution AFM imaging c rings reconstituted into 1-palmitoyl-2-oleoyl-sn-glyce- ro-3-phosphocholine at lipid-to-protein ratios of 0.5 and 1.0 assembled into 2D crystals that were exam- ined by high-resolution atomic force microscopy (AFM). AFM topographs revealed crystalline and paracrystalline membrane patches surrounded by the lipid bilayer (Fig. 2). c rings with an outer diameter of 5.8 ± 0.4 nm (n = 125) were surrounded by smal- ler c rings of diameter 5.4 ± 0.4 nm (n = 150). In agreement with previous measurements on c rings from other F 1 F 0 ATP synthases [21–23], the occur- rence of two diameters indicated that the reconsti- tuted c rings had an ‘upside-down’ orientation in the membrane and that we imaged both ring surfaces. In further agreement with previous measurements, the smaller c rings exhibited central protrusions that were shown to represent lipid headgroups [24] and to reflect the extracellular side of the ring [8,24]. Whereas the lipid bilayer exhibited a height of 4.5 ± 0.5 nm (n = 10), the proteins protruded 7.7 ± 0.5 nm (n = 10) from the supporting mica sur- face. At a lateral resolution of approximately 1 nm, the subunits of individual ring-shaped c oligomers became visible (Fig. 2A,B). Cross-correlation averages applied to further enhance common structural fea- tures showed different assemblies of the c rings, each being composed of 11 equally sized domains (Fig. 2C,D). Similarly, the reference-free averages generated by translational and rotational alignment of single c rings showed the same stoichiometry (Fig. 2D,E). From both the raw data and averages of c rings, it was clear that they were composed of 11 domains each, corresponding to 22 transmembrane helices. Structural investigations of the A. woodii c ring The same 2D crystals of the A. woodii c ring used for AFM were also used for structural investigations by electron microscopy. The A. woodii c ring sample con- sisted of vesicles containing crystalline areas with dimensions up to 0.5 lm. The 2D crystals are of plane group p22 1 2 1 (Fig. 3). The unit cell has dimen- sions of 100 · 108 A ˚ and contains four c rings, each with 11 densities. The crystals are tightly packed, and each ring is in contact with at least four neighbouring c rings. The projection map, calculated to 5 A ˚ resolu- tion (Fig. 3), shows that each c ring comprises two concentric, slightly staggered, packed rings, each com- posed of 11 densities. Whereas the inner ring of den- sities is tightly packed, the outer one is more loosely arranged, and the densities correspond to the N- and C-terminal helices of the c ring, respectively. The C-terminal helices show a clear handedness, and two of the rings face in the opposite direction in the membrane to the other two, forming the same pattern as in the AFM surface representation of Fig. 2A. By comparison with the 3D structure of the I. tartaricus c ring [24], the black rings represent the view from the cytoplasm (open rings in AFM) and the red ones the view from the extracellular side (smaller, closed rings in Fig. 3). The inner and outer diameters of the rings, measured at the density borders, are approxi- mately 17 and 50 A ˚ . However, the resolution obtained did not enable us to distinguish c 1 from c 2 ⁄ 3 . Subunit composition of the c ring from A. woodii The above structural analyses clearly assigned 22 transmembrane helices to the c ring of A. woodii.To unravel the c 1 and c 2 ⁄ 3 subunit composition of the potential hetero-oligomeric ring, we used laser-induced Fig. 2. High-resolution AFM topographs of reconstituted c rings. (A, B) Crystalline assemblies of c rings. Although the number of subunits forming the rings can be seen, the signal-to-noise ratio may be further enhanced by calculating their averages. (C, D) Nonsymmetrized correlation averages of both crystalline assem- blies reveal 11 masses forming the c ring. Each ring is neighbored by rings exposing either their wide or narrow ends. (E, F) Reference-free correlation averages for the two assemblies revealed 11 masses forming the wide and narrow ends of the rings. AFM topographs were recorded in dialysis buffer and exhib- ited gray levels correspond to a vertical scale of 3 nm. M. Fritz et al. Hybrid V 0 –F 0 rotor in a F 1 F 0 ATP synthase FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2001 liquid beam ion desorption mass spectroscopy (LIL- BID-MS), a recently established method to determine subunit stoichiometries in membrane protein com- plexes such as the cytochrome oxidase from Para- coccus denitrificans [25] and c rings from various organisms [26]. Figure 4 shows MS measurements of the A. woodii c ring purified from cells grown on fructose, taken under various desorption conditions. The mass spectrum in Fig. 4A shows an m ⁄ z distribu- tion of the complex with charges varying from 1 to 5. Individual peaks broadened towards higher masses, due to detergent and water molecules that stayed attached to the ring under the ultra-soft desorption process [27]. The overall mass of the c ring was deter- mined to be 93.5 ± 0.1 kDa. Harsher desorption con- ditions, achieved by increasing the laser intensity, led to the detachment of detergent and water molecules. Moreover, additional energy was transferred into the system, and the c rings (partly) dissociated into single subunits and subcomplexes. The mass spectrum in Fig. 4B was used to determine the c 1 to c 2 ⁄ 3 stoichio- metry. The peak distribution contains two series of subcomplexes. One series corresponds to subcomplex- es containing only c 2 ⁄ 3 units of the form (c 2 ⁄ 3 ) n , where n = 1–5, the other is built up from c 1 and c 2 ⁄ 3 units in the form c 1 (c 2 ⁄ 3 ) n , where n = 0–9. No sub- complexes that contain two or more c 1 subunits were detectable. The mass of the c 1 monomer was deter- mined to be 18.7 ± 0.1 kDa, and that for the c 2 ⁄ 3 monomer was 8.3 ± 0.1 kDa. Comparison of the spectra of the complete ring and the fragments revealed a stoichiometry for c 1 : c 2 ⁄ 3 of 1 : 9, leading to a mass of 93.4 kDa and hence 22 transmembrane helices for the c ring. Fig. 3. Electron microscopy of 2D crystals from A. woodii c rings. Projection map of 13 merged images at 5 A ˚ resolution. One unit cell of plane group p22 1 2 1 with its symmetry elements (two-fold rotation axes and screw axes) is indicated. The unit cell measures 100.3 · 108.5 A ˚ and contains four c rings. Hybrid V 0 –F 0 rotor in a F 1 F 0 ATP synthase M. Fritz et al. 2002 FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS Does the subunit composition of the c ring from A. woodii vary with the carbon source? As outlined above, the unique presence of a eukaryal V 0 -like c subunit in a bacterial ATP synthase raised the question whether the stoichiometry of V 0 :F 0 -like subunits may be flexible and thus a mechanism to change the action of the enzyme from ATP synthase to ATPase. To address potential variation in c ring subunit composition depending on the growth condi- tions, cells were grown under autotrophic conditions (ATP synthase required) or heterotrophic fermenting conditions (ion-pumping ATPase function required), and c rings were purified and subjected to LILBID analysis. The c rings of cells grown on fructose (20 mm), methanol (60 mm) or betaine (40 mm) (all heterotrophic) and on formate (80 mm) (autotrophic) showed an identical stoichiometry for c 1 : c 2 ⁄ 3 of 1 : 9, thus excluding the possibility of carbon source-depen- dent variation. Discussion A critical and long-standing question in (bacterial) bio- energetics is whether the ratio of translocated ions to ATP for a given ATP synthase is a fixed value. This value depends on the one hand on the number of cata- lytic sites, which seems to be invariable as all the enzymes analyzed so far have a a 3 b 3 (F 1 F 0 )orA 3 B 3 (A 1 A 0 ,V 1 V 0 ) stoichiometry [28]. The uncertainty lay in the number of ion-translocating subunits in the membrane-embedded rotor. Recently, the atomic struc- ture of a c ring from the F 1 F 0 ATP synthase from I tartaricus was solved and revealed 11 monomers [8]. Interestingly, on the basis of structural, biochemical and genetic studies, the c ring stoichiometry in F 1 F 0 ATP synthases is apparently variable among species. Ten monomers are found in c rings from the F 1 F 0 ATP synthases from yeast, Escherichia coli or Bacillus PS3 [29–31], undecameric rings were found in I. tar- taricus [22], Propionigenium modestum [17] and Clos- tridium paradoxum [32] Na + F 1 F 0 ATP synthases, a tridecameric c ring was found in the thermoalkaliphilic Bacillus sp. strain TA2.A1 [26], 14 subunits were found in the ATP synthase from spinach chloroplasts [21], and 13–15-meric c rings were identified in various cyanobacterial ATP synthases [23,33]. Less is known about the c subunit stoichiometries in the evolution- arily related V 1 V 0 ATPases, with only one structure solved, from Enterococcus hirae, which revealed 10 monomers [9]. The Na + F 1 F 0 ATP synthase operon of A. woodii is so far the only F 1 F 0 ATP synthase operon that has been found to encode F 0 and V 0 c subunit genes [14]. The genes have been found to be expressed [15] and the subunits have been found in the purified enzyme [16]. However, a critical question that was solved here was whether both subunits are part of one rotor or whether there are two populations of enzymes, one having only the F 0 -like c subunit and the other only Fig. 4. Mass spectra of the c ring taken under various laser desorption conditions. Under ultrasoft desorption conditions (A), the c ring is detected unfragmented with a charge distribution of one to four as indi- cated by red vertical bars. The broadening of the peaks towards higher masses is due to the attachment of detergent and water molecules. Under harsh desorption condi- tions (B), the c ring is fragmented, which leads to two series of subcomplexes con- taining only c 2 ⁄ 3 subunits (indicated by blue vertical bars) or one c 1 subunit and 1–9 c 2 ⁄ 3 subunits (indicated by red vertical bars). No subcomplex contains more than one c 1 sub- unit (theoretical masses of a c 1 series are indicated by green vertical bars). These find- ings and comparison of the two spectra reveal a c 1 : c 2 ⁄ 3 stoichiometry of 1 : 9 for the A. woodii c ring. M. Fritz et al. Hybrid V 0 –F 0 rotor in a F 1 F 0 ATP synthase FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2003 the V 0 -like c subunit. Here, we have unequivocally excluded the latter possibility. The LILBID analyses showed no peaks that contained two or more c 1 sub- units, excluding the existence of more than one c 1 unit per ring and of course rings formed from c 1 only. No mass was detectable corresponding to a ring made by c 2 ⁄ 3 subunits only or more than nine c 2 ⁄ 3 subunits. The stoichiometry of the subunits in the c ring of A. woodii was determined to be 1 : 9 (c 1 : c 2 ⁄ 3 ), with a total of 22 transmembrane helices. This value is identi- cal to the value obtained for the other Na + F 1 F 0 ATP synthases. Additionally, the size of the A. woodii c rings (approximately 58 A ˚ by AFM, approximately 50 A ˚ by electron microscopy) is comparable to those from I. tartaricus, P. modestum and C. paradoxum [17,32]. However, the major difference is that sub- unit c 1 not only lacks the conserved Na + binding site but also the essential negative charge (glutamate or aspartate) in transmembrane helix four as part of the ion-binding site. Therefore, the c ring of A. woodii has only 10 membrane-buried negative charges that are essential for binding the ion and also for the rotational mechanism of the ring. The c ring of I. tartaricus has 11 negative charges that are equally distributed along the horizontal axis of the rotor [8]. A positive charge on the stator attracts one of the negative charges on the ring and thus keeps the ring in place [34–36]. How- ever, the system is not stiff but instead the ring idles in front of the positive charge. This site is accessible to the outside, and ions (H + ,Na + ) flow from the outside to the binding site (driven by the electrical potential across the membrane) and occasionally bind to and thus compensate the negative charge on the rotor. The freed positive charge on the stator attracts the next negative charge on the rotor, thus leading to rotation of the ring. As most of the c rings investigated so far have a number of monomers that cannot be divided by three, this implies that the translocated ion to ATP ratio is not an integer. It has been suggested that an elastic power transmission between F 1 and F 0 is important for operation of the enzyme under symmetry mismatch conditions [37]. In the enzyme from A. woodii, rotation of the c ring over each phase of 120° is coupled to at least two different numbers of ions. Obviously, the force that has to be applied to overcome the spatial difference between three hairpins (c 2 ⁄ 3 ) –c 1,N-term ) c 1,C-term neutral – c 2 ⁄ 3 ) ) is more than that required to dis- locate just one hairpin. How this is achieved is unknown but is a challenging task for future studies. A variable number of identical c subunits in the ring was suggested to be a regulatory mechanism in E. coli [38], but this could not be confirmed experimentally in spinach chloroplast ATP synthase [39] or in the pres- ent study using the Na + F 1 F 0 ATP synthase from A. woodii. Rather, the stoichiometry seems to be fixed within a certain species and is determined by the geom- etry of the individual subunit [40–42]. The determined stoichiometry of 1 : 9 (c 1 : c 2 ⁄ 3 ) gives an Na + ⁄ ATP stoichiometry of 3.3, compared to 3.6 for the enzymes from P. modestum and I. tartaricus. It makes the enzyme from A. woodii a slightly better ATP-driven ion pump than an ATP synthase; whether this mar- ginal difference is of physiological relevance is ques- tionable but remains to be addressed experimentally. The electrochemical ion potential across the cyto- plasmic membrane of A. woodii has not yet been determined due to high nonspecific binding of the radioactive probes, but it is reasonable to assume that is similar to that in other bacteria, i.e. in the range of )180 to )200 mV. Therefore, the enzyme will work as an ATP synthase under physiological conditions. Its capability to synthesize ATP despite the presence of the V 0 -like c subunit has been demonstrated very recently in a proteoliposome system [16]. Experimental procedures Growth of cells and isolation of membranes A. woodii (DSM 1030) was grown in 20-1iter fermentors to mid-exponential growth phase as described previously [43]. Fructose (20 mm), betaine (40 mm), methanol (60 mm)or formate (80 mm) were used as carbon and energy sources. The NaCl concentration was 20 m m, unless otherwise stated. The ATP synthase was purified to apparent homo- geneity by solubilization with 1% dodecyl- b -d-maltoside followed by chromatography as described previously [16]. All preparations were routinely analyzed by SDS–PAGE using the buffer system described by Scha ¨ gger and von Jagow [44]. Polypeptides were visualized by staining with Coomassie brilliant blue [45] or silver [46]. The protein concentration of samples was determined according to the Lowry method [47], with BSA as a standard. Purification of c rings from A. woodii F 1 F 0 ATP synthases The ATP synthase from A. woodii was purified as previ- ously described [16], and the c ring was purified as described previously [17] with some modifications. The purified enzyme was incubated with 1.5% N-lauroylsarco- sine at 68 °C for 20 min. After cooling to 20 °C, (NH 4 ) 2 SO 4 was added to a saturation of 68%. After 2 h of incubation at 20 °C, the precipitated protein was removed Hybrid V 0 –F 0 rotor in a F 1 F 0 ATP synthase M. Fritz et al. 2004 FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS in a first step by filtration (filter paper, 2.6 lm pore size, Schleicher & Schuell, Dassel, Germany) followed by filtra- tion through a 0.2 lm filter (4 mm syringe filters, Nalgene, Rochester, NY, USA). The filtrate was dialyzed overnight at 4 °C against 10 mm Tris ⁄ HCl, 200 mm NaCl, pH 8.0, followed by addition of b-octylglycoside (Biomol, Mu ¨ n- chen, Germany) to a final concentration of 1.5%. To fur- ther concentrate the c rings and to remove excess salt, the sample was loaded onto an Amicon Ultra-4 tube (30 000 Da molecular mass cut-off; Amicon, Hanover, Germany) and concentrated to about 2–4 mgÆmL )1 . Western blot analysis After separation by SDS–PAGE, the ATP synthase subun- its were blotted onto a nitrocellulose membrane as described previously [48]. Western blot enhanced chemilu- minescence (ECL) detection reagents were either purchased from Perkin Elmer Life Sciences (Boston, MA, USA) or produced in our laboratory. Blot membranes were incu- bated in a mixture of 4 mL of solution A (0.1 m Tris ⁄ HCl, pH 6.8, 50 mg luminol in a total volume of 200 mL), 400 lL of solution B (11 mg p-hydroxycoumaric acid in 10 mL dimethylsulfoxide) and 1.2 lLofH 2 O 2 for 2 min before exposure to WICORex film (Typon Imaging AG, Burgdorf, Switzerland). Two-dimensional crystallization of the c ring For crystallization in 2D according to the method described previously [22], a sample of c ring (2 mgÆmL )1 ) was mixed with 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids Inc., Alabaster, AL, USA) at lipid-to- protein ratios of 0.5, 1.0 and 1.5 w ⁄ w. The mixture was dialyzed in 50 lL buttons (Hampton Research, Aliso Viejo, CA, USA) against 50 mL of 10 mm Tris ⁄ HCl (pH 8.0), 200 mm NaCl and 3 mm NaN 3 for 24 h at 25 °C and another 24 h at 37 ° C. The 2D crystals were stored at 4 °C until further analysis. Atomic force microscopy An atomic force microscope (Nanoscope IIIa; DI-Veeco, Santa Barbara, CA, USA), equipped with a 100 lm X–Y piezo scanner, was optimized for observing single molecules in the buffer solution. The 100 lm-long silicon nitride AFM cantilevers (Olympus, Tokyo, Japan) had nominal spring constants of 0.9 N ⁄ m. To adsorb the protein mem- branes, 20 mL of the sample buffer (approximately 10 lgÆmL )1 reconstituted c rings, 10 mm Tris ⁄ HCl, 200 mm NaCl, 0.02% NaN 3 , 10% glycerol, pH 7.8) was placed onto freshly cleaved mica for about 30 min. Then the sample was rinsed with dialysis buffer to remove weakly attached material. Contact-mode AFM topographs were recorded in dialysis buffer at 25 °C, with a loading force of approxi- mately 100 pN and a line frequency of 4–6 Hz. No differ- ences between topographs recorded in the trace and retrace directions were observed, indicating that the scanning pro- cess did not influence the appearance of the sample. For image processing, individual particles of the AFM topo- graphs were subjected to reference-free alignment and averaging using the SPIDER image processing system (Wadsworth Labs, New York, NY, USA). Correlation averages were calculated using the SEMPER image process- ing system (Synoptics Ltd, Cambridge, UK). To assess the rotor symmetry, the rotational power spectra of reference- free averages and of single rotors were calculated. Electron microscopy and image processing Two-dimensional crystal samples were prepared in 4.5% w ⁄ v trehalose on molybdenum grids (Pacific Grid-Tech, San Diego, CA, USA) by the back-injection method. Grids were examined in a JEOL 3000 SFF helium-cooled electron microscope (JEOL Ltd., Tokyo, Japan) at 4 K at an accel- erating voltage of 300 kV. Images were recorded by a spot- scanning procedure, using 24 spots by 30 spots per image on Kodak SO-163 film (Kodak, Stuttgart, Germany) at a magnification of 53 000 · and with an electron dose of 20– 30 electrons ⁄ A ˚ 2 . The films were developed for 12 min in full-strength Kodak D-19 developer. Images selected by optical diffraction were digitized on a Zeiss SCAI scanner (Zeiss, Jena, Germany) using a pixel size of 7 lm, corre- sponding to 1.3 A ˚ on the specimen. Images were processed using MRC [49] and CCP4 [50]. Data were merged to a resolution of 5 A ˚ . LILBID LILBID-MS [25,27] works with liquid sample targets. Therefore, microdroplets of the sample solution (50 lm diameter, volume 65 pL) were introduced into a vacuum using an on-demand droplet generator at a frequency of 10 Hz. The droplets are irradiated one by one by IR laser pulses, tuned to the absorption maximum of water at around 2.8 lm. The laser energy is transferred into the stretching vibrations of water, leading to a supercritical state of the liquid. The droplets explode and the charged biomolecules in the solution are set free. Those that escape the following charge neutralization are accelerated and mass-analyzed in a time-of-flight reflectron mass spectrome- ter constructed in our laboratory. Acknowledgements This work was supported by the Deutsche Forschungs- gemeinschaft (SFB 472 to VM and Werner Ku ¨ hlbrandt, M. Fritz et al. Hybrid V 0 –F 0 rotor in a F 1 F 0 ATP synthase FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2005 SFB 579 to BB, Cluster of Excellence ‘Macromolecular Complexes’ Project EXC 115 to TM), the Fonds der chemischen Industrie (to BB), and the EU (grant NEST2004 PathSYS29084 to DM). 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Hybrid V 0 –F 0 rotor in a F 1 F 0 ATP synthase FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2007 . An intermediate step in the evolution of ATPases – a hybrid F 0 –V 0 rotor in a bacterial Na + F 1 F 0 ATP synthase Michael Fritz 1, *, Adriana L membrane [ 4–6 ]. Subunit c of the F 1 F 0 ATP synthases has a molecular mass of approximately 8 kDa, and folds in the mem- brane like a hairpin, with two transmembrane helices connected