an intermediate step in the evolution of atpases – a hybrid f0–v0 rotor in a bacterial na+ f1f0...

An intermediate step in the evolution of ATPases – ahybrid F0–V0 rotor in a bacterial Na+ F1F0 ATP synthaseMichael Fritz1,*, Adriana L. Klyszejko2,*, Nina Morgner3,*, Janet Vonck4, Bernd Brutschy3,Daniel J. Muller2, Thomas Meier4 and Volker Muller1

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 F1F0 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 (F1)

and a membrane-embedded, ion gradient-driven motor

(F0) [3]. The membrane-embedded motor comprises a

stator and a rotor. The stator is formed by subunits a

and b2, 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 F1F0 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; F0-V0 hybrid rotor

Correspondence

V. Muller, 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: [email protected]

*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+ F1F0 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 F0-like c subunits (c2 and c3),

with two transmembrane helices, and a 18 kDa eukaryal V0-like (c1)

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 c2 ⁄ c3 : c1 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 F0–V0 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 (DlH+

⁄ Na+) of

around )200 mV and a phosphorylation potential

(DGp) of 60 kJÆmol)1 according to the equation:

DGp ¼ nFDlNaþ

where n is the number of translocated ions and F is

the Faraday constant.

The c subunit of the eukaryal V1V0 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 V0 and F0, the rotor of

eukaryal V1V0 ATPases has only half the number

of ion-binding sites compared to F1F0 ATP syntheses.

This low H+(Na+) ⁄ATP ratio is apparently the reason

for the inability of eukaryal V1V0 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 V1V0 ATPases is to energize endo-

cytoplasmatic membranes in the eukaryotic cell [13].

The Na+ F1F0 ATP synthase operon from the

anaerobic, acetogenic bacterium Acetobacterium woodii

differs from all other F1F0 ATP synthases by the

presence of one V0 subunit c gene (atpE1) and two

genes (atpE2 ⁄ atpE3) encoding identical F0 c subunits.

The gene atpE1 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 atpE2 and atpE3 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 V0–F0 hybrid rotor, the

first found in nature.

Results

Purification of the c ring from A. woodii

The c rings of the F1F0 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 c1 as well as c2 ⁄ 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

c1 : c2 ⁄ 3. As observed before for the c rings of entire

Na+ F1F0 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+ F1F0 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 c1 (lanes 7 and 8)

or subunit c2 ⁄ 3 (lane 9). The antibody

against c2 ⁄ 3 also reacts with c1.

Hybrid V0–F0 rotor in a F1F0 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 F1F0 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 p22121 (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 c1 from

c2 ⁄ 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 c1 and c2 ⁄ 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 V0–F0 rotor in a F1F0 ATP synthase


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 c1 to c2 ⁄ 3 stoichio-

metry. The peak distribution contains two series of

subcomplexes. One series corresponds to subcomplex-

es containing only c2 ⁄ 3 units of the form (c2 ⁄ 3)n,

where n = 1–5, the other is built up from c1 and c2 ⁄ 3

units in the form c1(c2 ⁄ 3)n, where n = 0–9. No sub-

complexes that contain two or more c1 subunits were

detectable. The mass of the c1 monomer was deter-

mined to be 18.7 ± 0.1 kDa, and that for the c2 ⁄ 3

monomer was 8.3 ± 0.1 kDa. Comparison of the

spectra of the complete ring and the fragments

revealed a stoichiometry for c1 : c2 ⁄ 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 p22121 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.



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

V0-like c subunit in a bacterial ATP synthase raised

the question whether the stoichiometry of V0 : F0-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 c1 : c2 ⁄ 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 a3b3 (F1F0) or A3B3

(A1A0, V1V0) 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 F1F0 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 F1F0

ATP synthases is apparently variable among species.

Ten monomers are found in c rings from the F1F0

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+ F1F0 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 V1V0 ATPases, with only one structure

solved, from Enterococcus hirae, which revealed 10

monomers [9].

The Na+ F1F0 ATP synthase operon of A. woodii is

so far the only F1F0 ATP synthase operon that has

been found to encode F0 and V0 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 F0-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 c2 ⁄ 3 subunits (indicated by blue

vertical bars) or one c1 subunit and 1–9 c2 ⁄ 3

subunits (indicated by red vertical bars). No

subcomplex contains more than one c1 sub-

unit (theoretical masses of a c1 series are

indicated by green vertical bars). These find-

ings and comparison of the two spectra

reveal a c1 : c2 ⁄ 3 stoichiometry of 1 : 9 for

the A. woodii c ring.



the V0-like c subunit. Here, we have unequivocally

excluded the latter possibility. The LILBID analyses

showed no peaks that contained two or more c1 sub-

units, excluding the existence of more than one

c1 unit per ring and of course rings formed from c1only. No mass was detectable corresponding to a ring

made by c2 ⁄ 3 subunits only or more than nine c2 ⁄ 3

subunits.

The stoichiometry of the subunits in the c ring of

A. woodii was determined to be 1 : 9 (c1 : c2 ⁄ 3), with a

total of 22 transmembrane helices. This value is identi-

cal to the value obtained for the other Na+ F1F0 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 c1 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 F1 and F0 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 (c2 ⁄ 3) – c1,N-term

)

c1,C-termneutral – c2 ⁄ 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+ F1F0 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 (c1 : c2 ⁄ 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 V0-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 mm, 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 Schagger 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 F1F0 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,(NH4)2SO4 was added to a saturation of 68%. After 2 h of

incubation at 20 �C, the precipitated protein was removed



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, Mun-

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 lL of H2O2 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 NaN3 for 24 h at 25 �C and

another 24 h at 37 �C. The 2D crystals were stored at 4 �Cuntil 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% NaN3, 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 ⁄ A2. 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 lmdiameter, 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 Kuhlbrandt,



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). We thank Deryck

Mills for assistance with electron microscopy and

Werner Kuhlbrandt (MPi of Biophysics, Frankfurt,

Germany) for comments on the manuscript.

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an intermediate step in the evolution of atpases – a hybrid f0–v0 rotor in a bacterial na+ f1f0...

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