Tài liệu Báo cáo khoa học: Rotary F1-ATPase Is the C-terminus of subunit c fixed or mobile? docx

Rotary F
1
-ATPase
Is the C-terminus of subunit c fixed or mobile?
Martin Mu¨ ller, Karin Gumbiowski, Dmitry A. Cherepanov, Stephanie Winkler, Wolfgang Junge,
Siegfried Engelbrecht and Oliver Pa¨ nke
Universita
¨
t Osnabru
¨
ck, FB Biologie/Chemie, Abt. Biophysik, Osnabru
¨
ck, Germany
F-ATP synthase synthesizes ATP at the expense of ion
motiveforcebyarotarycouplingmechanism.Acentral
shaft, subunit c, functionally connects the ion-d riven rotary
motor, F
O
, with the rotary chemical reactor, F
1
.Using
polarized spectrophotometry we have demonstrated previ-
ously the functional rotation of the C-terminal a-helical
portion of c in the supposed ‘hydrophobic bearing’ formed
by the (ab)
3
hexagon. In apparent contradiction with these
spectroscopic results, an engineered disulfide bridge between
the a-helix of c and subunit a did not impair enzyme activity.
Molecular dynamics simulations revealed the possibility of a
‘functional unwinding’ of the a-helix to form a swivel joint.

Furthermore, they suggested a firm clamping of that part of
c even without the engineered cross-link, i.e. in the wild-type
enzyme. He re, we rechecked the rotational mobility of the
C-terminal portion of c relative to (ab)
3
. Non-fluorescent,
engineered F
1
(aP280C/cA285C) was oxidized to form a
(nonfluorescent) ac heterodimer. In a second mutant,
containing just the point mutation within a, all subunits were
labelled with a fluorescent d ye. Following disassembly and
reassembly of the combined preparations and cystine
reduction, the enzyme was exposed to ATP or 5¢-adenylyl-
imidodiphosphate (AMP-PNP). After reoxidation, we
found fluorescent ac dimers in all cases in accordance with
rotary motion of the entire c subunit under these conditions.
Molecular dynamics simulations covering a time range of
nanoseconds therefore do not necessarily account for mo-
tional freedom in microseconds. The rotation of c within
hours is compatible with the spectroscopically detected
blockade of rotation in the AMP-PNP-inhibited enzyme in
the time-range of seconds.
Keywords: ATP hydrolysis; catalytic mechanism; F
1
-ATP-
ase; molecular dynamics calculation; motor protein.
F
O
F

1
-ATP synthase of bacteria, chloroplasts, and mito-
chondria catalyses the endergonic synthesis of adenosine
triphosphate (ATP) from adenosine d iphosphate (ADP)
and phosphate (P
i
) using a transmembrane proton-motive
or sodium-motive force. In reverse, F
O
F
1
is capable of
generating ion-motive force at the expense of ATP hydro-
lysis. The enzyme, in its simplest bacterial form (Escherichia
coli), consists of eight different subunits, a
3
b
3
cde in F
1
,the
catalytic headpiece, and ab
2
c
10
in F
O
, the ion-translocating
membrane portion. Energy is mechanically transferred
between F

O
and F
1
by rotation of the central shaft (cec
10
),
relative to the stator subunits (a
3
b
3
dab
2
). Both complexes,
F
O
and F
1
, are rotary steppers (for recent reviews, see [1–8]).
Based upon crystal structure analysis it has been hypo-
thesized [9] and later s hown by chemical cross-linking [10],
by polarized absorption recovery after photobleaching [11],
and most spectacularly by videomicroscopy [12–14], that
ATP hydrolysis by isolated and immobilized F
1
-ATPase
drives the rotation of the central shaft, subunit c,relativeto
the hexagon formed by subunits (ab)
3
. Portions of subunits
a and b provide a snug fit for the a-helical C-terminal

portion of c, considered to form a ‘hydrophobic bearing’
and to be essential for rotary function [9]. The functional
rotation of the penultimate amino acid at the C-terminus of
c relative to the immobilized remainder of chloroplast F
1
has b een detected by polarized photobleaching (with eosin
as probe) [11,15–17]. This finding was difficult to reconcile
with the observation that up to 12 amino acid residues could
be deleted by site-directed mutagenesis without suppressing
catalysis [18,19] or impairing c rotation [18] (Fig. 1). It was
even more difficult to reconcile with the lack of inhibition of
ATP h ydrolysis a nd c rotation after covalent disulfide-
bridging subunits a and c at positions aP280C and cA285C
[20] (Fig. 1). One way to interpret this finding was to assume
that the a-helix at the C-terminal portion of subunit c was
unwound to provide swivel joints a round one or several
dihedral angles, in other words, that c under these
conditions did not rotate in its entirety, but just in part.
Molecular dynamics simulations of ac cross-linked
enzyme revealed that the torque generated by the enzyme
is sufficient to u nwind the a-helix at the C-terminal portion
of c thus impelling the backbone rotation around Rama-
chandran dihedral angles [20]. Further calculations with the
noncross-linked enzyme suggested a firm clamping of the
C-terminal c portion within (ab)
3
(this work). This would
make the proposed unwinding of the a-helix in c afeatureof
the wild-type enzyme and an integral element of the catalytic
mechanism. Such a permanent immobilization of the

Correspondence to O. Pa
¨
nke, Universita
¨
t Osnabru
¨
ck, FB Biologie/
Chemie, Abt. Biophysik, Barbarastr.11, D-49076 Osnabru
¨
ck,
Germany. Fax: +49 541 969 2870, E-mail:
Abbreviations:F
O
, ion-driven rotary motor of F-ATP synthase; F
1
,
rotary chemical reactor of F-ATP synthase; AMP-PNP, 5¢-adenylyl-
imidodiphosphate; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid);
TMR-ITC, tetramethyl rhodamine-5-isothiocyanate.
(Received 30 April 2004, revised 30 July 2004, accepted 6 August 2004)
Eur. J. Biochem. 271, 3914–3922 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04328.x
C-terminal portion of c, however, would contradict previ-
ously pu blished spectroscopic results revealing its ATP-
dependent functional rotation [11,17].
We reassessed the rotational mobility of this portio n of c
with cleavable cross-links similar to t he approach used b y
Duncan et al. [10]. The experimental design is shown in
Fig. 2. By dissociation and reconstitution of appropriately
tailored F
1

complexes fluorescent a subunits were incor-
porated into the two noncross-linked positions of subunit a
of the oxidized mutant MM10 (Fig. 2). After reduction of
the nonfluorescent cross-linked ac the effects of ligand
binding and catalysis on the ability of the c-C-terminus to
reposition itself relative to specific a subunits was tested.
Upon reoxidation we found fluorescent ac dimers after
catalytic turnover or substrate binding, and even if the
enzyme was left without nucleotides and phosphate. The
formation o f a fluorescent ac cross-link could b e p revented
only by omitting the red uction/reoxidation cycles alto-
gether. T hese data reveal ed the C-terminal portion of
subunit c always t o be (rotary) mobile at the time scale of
this experiment, i.e. within hours.
Experimental procedures
Chemicals and enzymes
All restriction and DNA modifying enzymes were obtained
from New England Biolabs (Frankfurt/Main, Germany) or
Fig. 1. Schematic re pres entation o f E. coli F
1
and the calculated
unwinding of subunit c. (A) Localization of the engineered cysteine
residues within E. coli F
1
-mutant MM10. Two copies each of subunits
a and b are omitted for clarity. Subunit a and b areontheleftandthe
right side, respectively. Both point mutations, aP280C and cA285C,
are shown in black. The 12 amino acid residues shown in spacefill
representation can be truncated without inhibition of the rotary
mechanism [18]. Mutant MM6 has o ne cysteine, aP280C, only. The

residue coordinates were from a homology model constru cted previ-
ously [ 46]. (B ) Snapshots of c conformation during the f orced
molecular dynamics calculated with the torque of 56 pNÆnm. The
secondary structure of c is shown for the time of 1 ns (the end of initial
equilibration), 17 ns (half of turnover), 23 n s (one turnover) and 32 ns
(the end of final equilibration).
Fig. 2. Experimental flow-chart to test the rotational motion of the
C-terminal portion of subunit c. Su bunits a, b and c are shown as
circles, squares and triangles, respectively. Subunits shown in grey or
white are labelled or unlabelled, respec tively. Hatch ed subu nits are
either labelled or not as a result of the reassociation proc ess (see text
for details). Small black dots indicate the engineered cysteines aP280C
and cA285C.
Ó FEBS 2004 Rotation of subunit c in E. coli F
1
-ATPase (Eur. J. Biochem. 271) 3915
MBI Fermentas (St. Leon -Rot, Germany). Benzonase was
from Merck (Darmstadt, Germany). Oligonucleotide
primers were s ynthesized by MWG-Biotech (Ebersberg,
Germany). Nickel-nitrilotriacetic acid s uperflow was ob-
tained from Qiagen (Hilden, Germany) and tetramethyl-
rhodamine-5-isothiocyanate (TMR-ITC) was from
Molecular Probes ( Leiden, the Netherlands). All other
reagents used were of the highest grade c ommercially
available.
Strains and plasmids
The plasmids pMM10 (aP280C/cA285C) and pMM6
(aP280C) were generated as described by Gumbiowski
et al. [20]. In both cases plasmid pKH7 (all wild-type
cysteines substituted by alanine [21], His

6
-tag extension at
the N-terminus of subunit b, cK108C [13]) was used as
starting material. I n brief, the mutation cA285C was
generated by standard PCR with pKH7 as template DNA
and using KpnIandSacI for transferring the PCR product
into pKH7 (resulting in plasmid pMM9). The mutation
aP280C was generated using a method described by Weiner
et al. [22] with the subclone pMM3 [pBluescript II SK(+)
containing the KpnI/XhoI fragment of pKH7] as template
DNA. The exchange of the KpnI/XhoI fragment o f pKH7
with the corresponding fragment carrying the aP280C
mutation resulted in plasmid pMM6. Plasmid pMM10 w as
generated by replacement of the KpnI/SacI fragment of
pMM6 with the corresponding fragment of pMM9. E. coli
strains used were DH5a for plasmid preparation and DK8,
which contains a D(uncB-uncC) deletion [ 23], for expression
of E. coli F
1
.
Expression and purification of
E. coli
F
1
Preparation of F
1
was performed essentially as described
previously [18] except for the following modifications. Cells
were now h arvested at A
600

 1.8. Furth ermore the buffer
for resuspension of the cells a fter harvesting contained no
EDTA-free protease inhibitor mixture tablet. Instead, the
resuspended cells were incubated with ‡ 37 5 U Benzonase
per 100 mL for 15–20 min at room temperature before Ribi
press passage (Ribi Cell Fractionator, Model RF-1, Sorvall,
Langenselbold, Germany). After elution of F
1
from the
anion exchange column the solution was supplemented with
1m
M
MgATP and 2 m
M
dithiothreitol. Next, the protein
was p recipitated with 3.2
M
(NH
4
)
2
SO
4
andstoredat4°C.
The yield was 2.5–3.0 mg protein per litre of culture volume.
Cross-linking and labelling of
E. coli
F
1
For cross-linking of F

1
mutant MM10 (aP280C/cA285C)
 15 mg protein were purified from (NH
4
)
2
SO
4
and
dithiothreitol by gel fi ltration through PD-10 columns
(Amersham Biosciences, Freiburg, Germany), which were
equilibrated with 5 0 m
M
Tris/HCl, 50 m
M
KCl, 5 m
M
MgCl
2
, 10% (v/v) glycerol, pH 7.5 (buffer A). The eluate
was supplemented with 2 m
M
ATP and 100 l
M
5,5¢-
dithiobis(2-nitrobenzoic acid) (DTNB) and the samples
were incubated for 16 h at room temperature. The reaction
was stopped by addition of 20 m
M
N-ethylmaleimide

followed b y a 10 min i ncubation at room temperature.
The probe was purified by gel filtration through PD-10
columns, which were equilibrated with dissociation buffer
(50 m
M
Mes/NaOH, 1
M
LiCl, 5 m
M
ATP, 0.5 m
M
EDTA,
pH 6.1). For labelling of the F
1
mutant MM6 (aP280C)
with the fluorescent dye TMR-ITC  30 mg protein were
purified from (NH
4
)
2
SO
4
and dithiothreitol by gel filtration
through PD-10 columns, which were equilibrated w ith
100 m
M
HEPES/NaOH, 50 m
M
KCl, 5 m
M

MgCl
2
,
pH 8.5. After determination of the protein concentration
a 50-fold molar excess of TMR-ITC was added and then
incubated for 1 h at room temperature. Free dye was
removed by gel filtration through P D-10 columns with
dissociation buffer. The de gree of labelling was d etermined
by measuring the absorbance of the purified sample at the
absorbance maximum of TMR-ITC (k
max
¼ 555 nm, e ¼
65000 cm
)1
Æ
M
)1
). The degree of labelling was usually
between 20 and 30 fluorescent dyes per F
1
-MM6.
Dissociation of
E. coli
F
1
mutants and reconstitution
of hybrid-F
1
Dissociation and reconstitution of F
1

was performed
essentially as described previously [10,24]. After oxidation
of F
1
-MM10 and dye labelling of F
1
-MM6, the two F
1
mutants were gel filtrated against dissociation buffer (see
above), m ixed in a ratio of 1 : 2 ( 10 mg F
1
-MM10 and
 20 mg F
1
-MM6) and frozen in liquid nitrogen. The
samples were thawed at room temperature and again frozen
inliquidnitrogen,andthenstoredat)80 °C. After thawing
at room temperature the dissociated samples were diluted to
0.5 mgÆmL
)1
protein concentration with reconstitution
buffer [50 m
M
Mes/NaOH, 10% (v/v) glycerol, pH 6.0]
and then dialyzed (SpectraPor, Spectrum Laboratories Inc.,
Rancho Dominguez, CA, USA) against reconstitution
buffer containing 2.5 m
M
MgATP for 16 h at room
temperature.

Reduction and reoxidation of hybrid-F
1
Reconstituted hybrid-F
1
was purified by nickel-nitrilotri-
acetic acid affinity chromatography. Columns were equil-
ibrated with reconstitution buffer containing 2.5 m
M
MgATP and bound product was washed with buffer A
containing 20 m
M
imidazole. After elution of purified
hybrid-F
1
with buffer A containing 150 m
M
imidazole
(yielding  1.5 mg of protein per 3 mL eluate) the degree of
labelling was determined as described above. Typical values
for the degree of labe lling were 1–4 fluorescent dyes per
hybrid-F
1
. Aliquots of reconstituted F
1
samples were
treated either w ith no nucleotide, with 4 m
M
5¢-adenylyl-
imidodiphosphate (AMP-PNP), with 4 m
M

AMP-PNP and
4m
M
ADP, or with 4 m
M
ATP. The samples were then
reduced by addition of 20 m
M
dithiothreitol and incubation
for 16 h at room temperature. After another additio n of
20 m
M
dithiothreitol and incubation for further 2 h the
samples were purified by gel filtration through NAP-10
columns (Amersham Biosc iences) and nucleotides were
added as d escribed above. The f ollowing gel filtration
buffers were used: (a) buffer A for the sample, which
contained no nucleotide and the sample, which contained
4m
M
ATP, (b) buffer A + 1 m
M
AMP-PNP for the
sample, which contained 4 m
M
AMP-PNP and (c) buffer A
3916 M. Mu
¨
ller et al.(Eur. J. Biochem. 271) Ó FEBS 2004
+1m

M
AMP-PNP and 1 m
M
ADP for the sample, which
contained 4 m
M
AMP-PNP and 4 m
M
ADP. The samples
were incubated for 2 h at room temperature and reoxidized
by a two-fold successive addition of 100 l
M
DTNB
followed by incubation at room temperature for 16 h and
2 h , respectively. The reaction was stopped by addition of
20 m
M
N-ethylmaleimide and incubation for 10 min at
room temperature. Samples were purified by gel filtration
through NAP-10 columns, which were equilibrated with
buffer A. After each reaction/purification step aliquots were
taken for determination of ATP hydrolysis activity, protein
concentration and for SDS/PAGE.
Molecular dynamics calculations
A three-dimensional model of the ‘hydrophobic b earing’ at
the C-terminal portion of c was built using the X-ray
structure of bovine enzyme (PDB entry 1E79 [25]). The
rotary shaft is comprised of 24 residues from the N-end of c
subunit (cA1–cK24) and 43 residues from its C-end (cT230–
cL272). The chosen portion included a major part of the

coiled-coil region of c and the complete a-helical C-terminus
as held within the top of (ab)
3
. The rotary axis z was aligned
along the main axis of the shaft. The ‘bearing’ included a
total of 138 residues from the neighbouring portion of (ab)
3
located within 1.8 nm from the rotary axis (70 residues
belonged to a and 68 residues to b). Hydrogen atoms and
terminal groups of the protein backbone w ere built by the
program
CHARMM
22 [26]. The protein was ‘solvated’ by
TIP3 rigid water molecules [27], which formed a cylinder
with diameter of 3.6 nm and height of 7.6 nm. In total, the
system contained 2952 protein atoms a nd 1104 water
molecules. The molecular dynamics simulations were per-
formed with the program
NAMD
2 [28] using the all-atom
empirical force field
CHARMM
22 [26]. A harmonic boundary
potential was applied to prevent water evaporation outside
the cylinder considered above. The backbone atoms of the
‘bearing’ were constrained at their crystallographic posi-
tions, while other protein atoms were unconstrained. The
electrostatic interactio ns w ere truncated by a cut-off
distance of 1.2 nm. The s ystem was equilibrated during
1 n s, and then the rotation of c was forced by a constant

torque applied t o the coiled-coil portion of c at the level of
cK18–cK21 and cD233–cS236 residues. The t orque was
created by external forces acting on the two groups of four
carbon atoms each. The first group included the C
a
atoms of
cK18, cI19, cT20 and cK21, and the second group C
a
atoms of cD233, cN234, cA235 and cS236. The magnitude
and direction of the forces was calculated at every step of the
molecular dynamics integration (1 fs step width) by a Tcl
script () using the current position of the
geometrical center of each group relative to the z-axis.
Ab initio
quantum chemistry calculations
These calculations were carried out within the limits o f the
ab initio Hartree–Fock method in the 6 –311++G basis set
using the
GAMESS
program complex [29]. The model s ystem
included a n A la-Gly dipeptide in t he neutral state with an
amidated C-terminus. The equilibrium configuration of this
dipeptide was obtained by the geometrical optimization in
the molecular mechanics force field, followed by semi-
empirical AM1 minimization , and finall y b y ab initio
minimization in the 6–311++G basis set. The potential
energy profiles along w and / dihedral coordinates were
calculated by the rotation of the dipeptide in discrete
equidistant 15° steps with the subsequent complete geom-
etry opt imization i n t he 6–311++G basis at the fixed

values for w or /, respectively. The potential energy of the
optimized structure was calculated in the 6–311++G basis
set using the second-order Mo
¨
ller–Plesset c onfiguration-
based correlation method [30].
Other procedures
ATP hydrolysis activity w as measured by determination of
released P
i
after incubation of the enzyme for 5 min at 37 °C
in a reaction mixture containing 50 m
M
Tris/HCl, 3 m
M
MgCl
2
,10m
M
NaATP, pH 8.0. The blue-coloured phos-
phomolybdate complex was photometrically detected at a
wavelength of 745 nm [31]. SDS/PAGE was carried out in
the Amersham Biosciences Phast system (Amersham Bio-
sciences) without 2-mercaptoethanol in the sample buffer.
Gels were stained with Coomassie Brilliant Blue R-250 [32]
and s ilver [33]. P rotein determinations were carried out
according to the method of Sedmak & Grossberg [34].
Results
In the two E. coli F
1

mutants, MM10 and MM6, u sed in
this study, all wild-type c ysteines were substituted b y
alanines, one novel cysteine in c (K108C) was introduced
and a His
6
-tag at the N-terminus of subunit b was added
[13]. MM10 contained t wo additional cysteines in positions
aP280C and cA285C, and was capable of forming a cross-
link upon oxidation with a yield of more than 98% [20].
Mutant MM6 contained only one additional cysteine in
position aP280C. In E. coli strain DK8 both mutants grew
on succinate as well as t he control (KH7 [13]). After
isolation and purification, ATPase activities under r educing
conditions were in the range of 130–160 UÆmg
)1
for both
mutants, without noticeable amounts of c ross-linked ac
(Fig. 3 , lanes 1 & 3). Figure 2 summarizes the protocol used
to test the rotational mobility of the C-terminal portion of
subunit c.
Cross-link formation and labelling
Mutant MM10 showed f ormation of an ac heterodimer
upon oxidation with DTNB. After 16 h incubation, th e c
monomer had disap peared completely, as checked by SDS/
PAGE(Fig.3,lane1&2).MM6failedtodoso,as
expected (Fig. 3, lane 3 & 4). Despite the cross-link MM10
showed normal ATP hydrolysis activities and c rota tion
[20]. This was previously interpreted such that the torque
generated b y ATP hydrolysis is sufficient to uncoil the
a-helix in the C-terminal portion of subunit c [20].

MM10 served as source for nonfluorescent ac hetero-
dimers. For the incorporation of fluorescent a subunits into
the two nonfluorescent a positions within F
1
,mutantMM6
was labelled with the amine-reactive fluorescent dye TMR-
ITC. Conditions were chosen to ensure a labelling by 20–30
TMR molecules per molecule of MM6. As shown in Fig. 3,
lane 5, the l abelling affected all five F
1
subunits. An
Ó FEBS 2004 Rotation of subunit c in E. coli F
1
-ATPase (Eur. J. Biochem. 271) 3917
additional band of high molecular mass as apparent in the
SDS gel was shown by Western blotting to consist of a
subunits only, but not c [20]; probably an aa homodimer.
It should be noted that MM6 could not form an ac
heterodimer because it lacked the essential c ysteine residue
(cA285C;Fig.3,lane4).
Reconstitution of hybrid-F
1
Labelled MM6 and cross-linked MM10 were dissociated by
a freeze-thaw proc edure in the presence of 1
M
LiCl
according to [ 10,24]. The sample s were m ixed at a ratio of
2 : 1 (MM6/MM10), dissociated, diluted and dialyzed
against reconstitution buffer containing 2.5 m
M

MgATP.
By application of nickel-nitrilotriacetic acid affinity chro-
matography, we obtained a solution containing a mixture of
labelled hybrid-F
1
along with unknown amounts of His
6
-
tagged b.Startingwith30mgofF
1
, about 1.5 mg protein
were obtained from the nickel-nitrilotriacetic acid column,
i.e. 5%. Assuming a homogeneous hybrid-F
1
preparation,
labelling ratios of 1–4 fluorescent dye molecules per F
1
were
determined. Two types of hybrid-F
1
species were expected,
depending on the origin o f c. O ne population of F
1
complexes was expected to contain nonfluorescent ac
heterodimers originating f rom mutant M M10, whereas
the second type should contain fluorescently labelled c from
MM6. Both types were expected to contain both fluorescent
and nonfluorescent subunits a and b (Fig. 2 ). The latter type
was unimportant in this context, as these enzymes lacked
the capability to form fluore scent ac cross-links, due to the

absence of the point mutation cA285C. Figure 3, lane 6,
shows t he result of the SDS/PAGE of the hybrid-F
1
preparation. The absence of c monomers in the SDS gel
indicated that the first type of hybrid-F
1
molecules,
containing nonfluorescent ac heterodimers, was formed
exclusively during the reconstitution procedure. The reason
for the absence of hybrid-F
1
containing fluorescent subunit
c from MM6 is unknown. Possibly, attached TMR
molecules prevented the formation of reassembled e nzymes
due to steric hindrance. Fluorescent a subunits were present
in hybrid-F
1
molecules, as was evident from the fluorescence
image of the SDS gel (Fig. 3, lane 6). The ab band, as well as
the aa homodimer band, were fluorescent. The activities of
hybrid-F
1
were dependent on the resulting labelling ratio.
Preparations with labelling ratios of about one dye molecule
per protein molecule had ac tivities of 110 UÆmg
)1
,which
were close to the original activities of the mutants MM10
and MM6 (130–160 UÆmg
)1

). At ratios of about four the
activity was about 40 UÆmg
)1
. This decrease, however, was
probably not only caused by the fluorescent dye, but also by
the presence of nonfunctional reassembled enzyme and
single b subunits.
Rotational mobility of the C-terminal portion of subunit c
Hybrid-F
1
, which contained the nonfluorescen t ac cross-
link, was expected to reveal fluorescent ac heterodimers
after reduction of the disulfide bridge, followed by rotation
of c upon ATP hydrolysis and subsequent reformation of
the disulfide bridge. To this end, aliquots of reconstituted F
1
samples were exposed to (a) no nucleotide at all, (b) AMP-
PNP, (c) AMP-PNP and ADP, or (d) ATP. Samples w ere
reduced by addition of dithiothreitol followed by gel
filtration in the presence of the respective substrate.
Afterwards, the disulfide bridge was r eformed by addition
of DTNB. After each reaction/purification step s amples
were taken for determination of ATP hydrolysis activity and
SDS/PAGE.
Table 1 summarizes the activities of a ll samples. In order
to compare the values fro m different expe riments with
different labe lling ratios the activities were normalized with
respect to the activity of the primary nickel-nitrilotriacetic
acid eluate. The relative activities remained unchanged
during the whole reduction/reoxidation procedure. The high

activity of the oxidized s amples and the quantitative c ross-
linking of subunit c with a (SDSgelinFig.3,lanes
8,10,12,14) suggested the unwinding of the a-helix of
subunit c in hybrid-F
1
molecules, as seen before with
MM10 [20]. The lack of inhibition by AMP-PNP i s
Fig. 3. SDS/PAGE (A) and the corresponding fluorescence images (B).
An 8–25% gradient gel (Amersham Biosystems Phast system) with 2%
(w/v) SDS was used and stained with Coomassie Brilliant Blue R-250
[32] followed by silver [33]. The protein concentration was 3 mgÆmL
)1
;
each lane con tained 0 .9 lg protein. Lanes 1–5 show the starting
material, F
1
mutants MM10 and MM6, in the reduced and oxidized
state as indicated. MM6 in lane 5 was labelled with 24 dye molecules.
Lane 6 was the hybrid-F
1
preparation before substrate incubation.
Lanes 7 –14 show the reduced and reoxidized e nzyme under di fferent
substrate conditions, as indicated. T he nucleotide concentration was
4m
M
for all nucleotides, throughout. Lane 15 shows hybrid-F
1
,which
was handled like the other samples but w as never reduced (control).
The labelling ratio of hybrid-F

1
was 1–4 TMR m olecules per F
1
.
3918 M. Mu
¨
ller et al.(Eur. J. Biochem. 271) Ó FEBS 2004
understandable, because the samples were diluted during
the activity a ssay and a dded ATP displaced the residual
amounts of AMP-PNP and ADP from the catalytic sites of
the enzyme. Activity measurements in the presence of 1 m
M
AMP-PNP or a mixture of 1 m
M
AMP-PNP and 1 m
M
ADP showed complete inhibition.
Figure 3 shows the corresponding SDS/PAGE analysis
ofthesamplesaftereachreactionstepandTable2
summarizes the fluorescence intensities of the correspond-
ing g el bands. After reduction of the hybrid-F
1
preparation
a c monomer band became clearly visible and a minor
amount of ac heterodimers was not reduced (Fig. 3 , lanes
7,9,11,13). As expected, the re oxidation of the samples
intensified the ac bands again and the c bands disappeared
completely (lanes 8,1 0,12,14). At the same t ime the ac
heterodimers showed fluorescence, consistent with a rota-
tional movement of the C-terminal portion of subunit c.

This behaviour was independent of the applied substrate
conditions. Even with AMP-PNP and a mixture of AMP-
PNP and ADP a fluorescent ac band was observed. This
was surprising because the ATP analogue AMP-PNP is
known to stabilize F
1
complexes and was added to
crystallization media in X-ray structure analysis [9,35–38].
To exclude that the fluorescent ac heterodimer was formed
due to impurities (e.g. nonreconstituted subunits) a control
sample was treated like the other samples with respect to
incubation times, gel filtration, etc., but without being
reduced. This c ontrol sample s howed a strong ac ban d in
the SDS gel, but no fluorescence (Fig. 3, l ane 15). We
checked whether or not a fl uoresc ent ac dimer was formed
after reduction due to a continuous disassembly/assembly
mechanism of F
1
molecules. For this purpose we labelled
wild-type-F
1
enzymes (BWU13 [39]), with TMR-ITC and
mixed them with an unlabelled F
1
mutant, KH7, carrying a
His
6
-tag at the N-terminus of subunit b [13]. After
overnight incubation, both mutants were separated by
nickel-nitrilotriacetic acid chromatography. The eluted

His
6
-tagged F
1
remained nonfluorescent ( < 3%), thus
excluding any interchange of subunits. Nevertheless, it was
apparent that the fluorescence intensity of the ac bands in
all reoxidized samples was rather weak, a lthough the SDS
band was very intense. This was not surprising, because
not all i nserted a and b subunits were labelled and only a
maximum of two-thirds of all ac heterodimers could have
contained a fluorescent a subunit. In fact, our results show
that about 14% of the total intensity was located in the ac
band (Table 2).
Molecular dynamics simulations of the rotary mobility of
c within the ‘hydrophobic bearing’ at the top of a
3
b
3
The molecular model of the rotary part of c and the
surrounding part of (ab)
3
was constructed as described in
Experimental procedures using available model coordinates
[9]. Unlike p revious simulations with the a-helical terminus
of subunit c from E. coli, the present simulations included a
large portion of the ‘hydrophobic bearing’ at the top of a
3
b
3

and the rotation of c was restricted only by steric
interactions with this hydrophobic collar. The system was
equilibrated for 1 n s, after that a rotary motion of c was
forced by a constant torque applied to its coiled-coil portion
at the l evel of cK18–cK21 and cD233–cS236. The simula-
tions included two traces obtained with the applied torque
of 56 and 112 pNÆnm, respectively (the average torque
generated by the enzyme at physiological conditions has
been found to be as high as 56 p NÆnm [40]). The angular
displacement of c as a function of time (calculated at the
level where the torque was applied) is shown in Fig. 4. The
right curve in this figure was obtained with a torque of
56 pNÆnm and the left one with a torque of 112 pNÆnm, the
arrows show the beginning (t ¼ 1ns)andtheend(t¼
23 ns) of the forced rotation. With 56 pNÆnm torque, the
relaxation of the system was calculated during the last 8 ns
of the dynamics. In both cases the a pplied torque caused a
complete unfolding of the single-helical portion of c at the
level of cR254–cV257 residues, a partial deformation of the
double-helical part of c, but the C-terminal portion of c was
tightly clamped within (ab)
3
and k ept its initial c onforma-
tion and orientation.
Table 1. Normalized activities of hybrid-F
1
preparations. After recon-
stitution a nd purification the hybrid-F
1
preparations had activities

between 40 and 110 UÆmg
)1
, depending on the resulting labelling ratios
(dye/protein), which had values between 4 and 1, respectively. In order
to compare different experiments with different dye contents the
activities were normalized to 100 with respect to the activity of the
primary nickel-nitrilotriacetic acid eluate.
Substrate for incubation
None ATP AMP-PNP AMP-PNP +ADP
Nickel-nitrilotriacetic
acid eluate
100 100 100 100
Reduced 120 149 102 112
Reoxidized 113 134 119 104
Table 2. Fluorescence intensities of the SDS gel bands. The fluorescence shown in Fig. 3 was analyzed with the
GELPRO ANALYSER
software from
Media Cybernetics (Silver Spring, MD, USA). The band intensities were baseline corrected and normalized to 100 with respect to the total intensity
of all bands in each sample lane. Red, reduced; Reox, reoxidized: Ox, oxidized.
Band
Substrate for incubation
None ATP AMP-PNP AMP-PNP + ADP Control
Red Reox Red Reox Red Reox Red Reox Ox
a
2
22 54 23 3 320
ac 2 17 2 12 3 12 2 14 2
ab 84 73 81 80 79 70 90 79 73
Ó FEBS 2004 Rotation of subunit c in E. coli F
1

-ATPase (Eur. J. Biochem. 271) 3919
With 56 pNÆnm torque, the secondary structure was
stable during the first 10 ns, after that a partial unfolding
began by a rotation of the peptide backbone between cA256
and cV257 residues. The initial conformational transition
included a simultaneous shift of the Ramachandran angle w
of cA256 by +120° and of the angle / of cV257 by )90°.
After t his initial unfolding event, fur ther unfolding
occurred, mainly due to rotation around w angles of
cR254, cA256, cQ255 and cV257 residues. At the end of
forced dynamics, the angle w of cA256 made a full turnover
by +360°. When the external torque was switched off at
t ¼ 23 ns, the warped double-helical part of c underwent an
elastic relaxation back to its initial outstretched conforma-
tion, whereas the conformation of cR254–cV257 residues
remained uncoiled.
When a twofold higher torque of 112 pNÆnm was
applied, the secondary structure a-helix was broken after
2.5 n s of the forced dynamics, the initial unfolding event
included almost simultaneous changes of Ramachandran
angles w of cR254 and cA256 by +120° and angles / of
cQ255 and cV257 by )90°. The further rotation of c was
caused mainly by rotation around w-angles of cR252,
cT253, cA256 and cV257 residues. The residues cR252 and
cT253 made more than one turnover around w-angle.
In both cases the m olecular dynamics simulations revealed
that rotation around the w Ramachandran angle w as
preferred over that around /. We calculated the potential
barrier f or the rotation around w and / angles in the
dipeptide Ala-Gly by ab initio quantum chemistry (program

GAMESS
[29] using Pople’s 6–311++G basis set and the
second-order Mo
¨
ller–Plesset configuration-based correla-
tion method). The potential barriers for the rotation a long
the wand /dihedral angles were 30 and 3 8 kJÆmol
)1
, respect-
ively. These values were about 25% higher than the figures
obtained by the molecular mechanics calculations [20].
The calculations indicated that in the crystallographic
structure the C-terminal portion of c seems to b e tightly
clamped within the ‘hydrophobic bearing’ at the top of
(ab )
3
. The steric constraints on the c rotation in this region
were essentially bigger than the rigidity of the single a-helix.
The secondary structure o f t he latter c ould be e asily
unfolded when the operational torque of 56 pN Ænm was
applied to the rotary shaft. At this magnitude of torque the
rotation around Ramachandran angles in the region o f
residues cT253–cV257 (cA267–cS271 in E. coli F
1
[41])
proceeded with a rate of 10
8
s
)1
, four orders of magnitude

faster than the observed r otary transitions in the enzyme
[42]. Because the molecular dynamics simulations were
performed with the frozen tertiary conformation of (ab)
3
,it
remained unclear whether large-scale fluctuations of the
(ab )
3
structure can open the ‘bearing’ at the time scale o f ls
to ms that is required for a rotation of c as a whole within
(ab )
3.
Discussion
This study was motivated by the previous finding that a
mutant F
1
(MM10) with a disulfide bridge engineered
between the stator subunit a and the C-terminal portion of
the rotary shaft, subunit c, showed unimpaired ATPase
activity and full torque in the videomicroscopy assay for
rotation [20].
The robustness of the bearing collar of (ab)
3
and the
rotational shaft, c, has been demonstrated by other
approaches: up to 12 amino re sidues at t he C-terminal
portion of subunit c were dispensable for catalysis and
rotation [18,19]. The C-terminus could be extended by
16 amino acid residues without drastic consequences
(a frameshift acco mpanied by b suppressor mutations)

[43]. Gre en fluo rescent p rotein could be fused to the
C-terminus of c without loss of enzyme function [44].
The crystal structure clearly pointed to limited freedom of
c to rotate other than around its long (‘vertical’) axis
(original suggestion by Abrahams et al.[9])andinthe
‘hydrophobic bearing’ formed by subunits a and b around
the C-terminal portion of c. Molecular dynamics calcula-
tions ([20] and this work) suggested the unwinding of the
single a-helix at the C-terminal portion of c, thus allowing
for unimpaired rotation of the remainder of c.Furthermore,
the calculations suggested the very end of c to be clamped
within the N-terminal collar of subunits ( ab)
3
permanently
and even without a disulfide bridge (this work), in seeming
contradiction with previous work employing the polarized
photobleaching of eosin [11,17].
The d ata presented here are clearly indicative of a
movement of the C-terminal portion of subunit c relative to
(ab )
3
within the time domain investigated, because the
originally cross-linked ac heterodimer consisted only of
nonfluorescent polypeptides, whereas after reduction/reoxi-
dation the r espective band contained fluorescent a.More-
over, the appearance of the fluorescent b and was not
dependent on conditions allowing for ATP hydrolysis.
The protocol we used did not (as with the original one
[10]) allow discrimination between translational o r rota-
tional movement of c. Because the microvideographic data

[12], however, clearly demonstrated unidirectional c rota-
tion upon ATP hydrolysis (even in the millisecond time
Fig. 4. Forced ro tation of c within (ab)
3
. The molecular model included
the central a-helical shaft (it covered the 3.2 nm long single-helical and
the 3.2 nm long double-coiled portions of c) and 138 residues of (ab)
3
located within a 7.6 nm long cylinder at the distance up to 1.8 nm from
the rotation axis. The constant torque of 56 and 112 pNÆnm (the
steeper and flatter curves, respectively) was applied to the two groups
of four C
a
atoms located on e turn abo ve the low er end o f c.The
arrows indicate the time interval of forced dynamics, after that the
system relaxation was monitored during 8 ns.
3920 M. Mu
¨
ller et al.(Eur. J. Biochem. 271) Ó FEBS 2004
range [42]), we interpret our findings also as indicative for
rotation, not just translation, even though s ome d ata
reported in the literature are still not compatible with the
concept of rotational catalysis [45].
Whether t he same holds true for t he AMP-PNP experi-
ment is more difficult to decide. Compared to the results of
Duncan et al. [10], where the corresponding disulfide bridge
was located close to the DELSEED sequence (bD380C/
cC87), we found fluorescent ac heterodimers even without
catalytic activity and in the p resence of AMP-PNP. T his
finding is in contrast with our previous observation b y

polarized photobleaching [11,17], which revealed blockage
of the functional rotation of c in some milliseconds by
added AMP-PNP, but it agrees well with the dat a of
Duncan et al.[10],wherec was allowed to rotate for about
10 min. In the presence of ATP, they observed an increased
amount of radiolabelled bc dimers, compatible with c
rotation. The yield of radioactive bc was decreased to about
30% upon inhibition or in the a bsence of ATP, but not to
zero (Figs 3 and 4 in [ 10]). In other words, i n t hese
experiments also, movement of c could not be blocked
entirely over a long time span.
Movement of c was probably possible within the time
scale of hours dictated in our approach by the required
protocol, as AMP-PNP might have been dissociated and
rebound occasionally. Whether c under these conditions
was able to carry out full rotation remains an open question,
at least its rotational o r translational freedom sufficed to
allow interaction with another a subunit than the one it was
connected to originally. An inspection of the X-ray struc-
ture, however, raises serious doubts about whether just a
‘bending’ movement of this portion of c might occur at all
and also at the same time be sufficient to induce the
observed cross-link. A rotational movement, this time
perhaps only around 120° and without preferential direc-
tion, thus would seem more plausible.
Why has the molecular dynamics calculation produced a
different result? Ignoring the limited section of the enz yme
that entered into the calculations and the fixed backbone at
the N-terminal portion of a and b, a simulation covering
some nanoseconds still cannot acco unt for domain flexibil-

ity in the range of microseconds. Evidence for such
fluctuations was obtained in our previous studies. The
rotational relaxation of the eosin attached to the C-terminal
portion of chloroplast c had components in the nanosecond
time range, but also another one at 30 ls (Fig. 4 in [17]).
These components have been interpreted to reveal the
librational motion of the dye molecule in narrow constraints
(ns) and subsequently in wider constraints by fluctuations
(30 ls) o f the N-terminal collar of (ab)
3
. The different
methodological approaches, the time domains they apply
to, and the associated mobility of subunit c are summarized
in Fig. 5.
The emerging picture is that the central shaft of F
1
never
comes to a full halt; c is free to slowly rotate back and forth
in the time range of minutes to hours, owing to the exchange
of bound substrates or inhibitors. ATP hydrolysis, on the
other hand, causes rapid unidirectional rotation in millisec-
onds and predominates so that futile mobility escapes
detection in activity a ssays. Solely in the time range of
nanoseconds the C-terminal portion might be permanently
clamped as proposed by the molecular dynamics calcula-
tions. A remarkable exception is the cross-linked mutant
MM10, where ATP hydrolysis-induced rotation overcomes
the artificial clamping of the C-terminal portion probably
by unwinding the a-helix to form a swivel joint.
Acknowledgements

Skillful technical assistance by Gabriele Hikade and Hella Kenneweg is
gratefully acknowledged. This work was supported by grants from the
DFG (SFB 431/P1) to W.J. and S.E., by the HSFP to W .J., by the
Volkswagenstiftung to W.J. and O.P., and the Fonds der Chemischen
Industrie to W.J.
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