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Conformation of rotating F1-ATPase revealed by single pair
fluorescence resonance energy transfer
Ryohei Yasuda1, Kengo Adachi2, Tomoko Masaike3, Hiroyuki Noji4,
Hiroyasu Itoh25, Masasuke Yoshida3, Kazuhiko Kinosita Jr26
1
Cold spring harbor laboratory, Cold spring harbor, NY11724 U.S.A.
2
CREST "Genetic Programming" Team 13, Teikyo Biotechnology Research Center
Nogawa 907, Kawasaki 216-0001, Japan
3
Research Laboratories of Resources Utilization, Tokyo Institute of Technology
Yokohama 226-8522, Japan
4
University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8505, Japan
5
Hamamatsu Photonics KK, Tokodai, Tsukuba 300-2635, Japan
6
Center for Integrative Bioscience, Okazaki National Research Institutes
Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan
To
whom correspondence should be addressed; E-mail: [email protected]
F1-ATPase is an ATP-driven rotary motor in which a rod-shaped  subunit rotates
inside a cylinder made of 33 subunits. To elucidate the conformation of rotating F1, we
measured fluorescence resonance energy transfer (FRET) between a donor on one of
three  and acceptor on  of a single F1 molecule. The yield of FRET changed stepwisely
at low [ATP] (1 M), reflecting the stepwise rotation of . The rotation angle of 
relative to the 33 cylinder on an ATP-waiting state was obtained from the FRET yield,
and was consistent with crystal structures of mitochondrial F1 (MF1).
F1 is one of two units of an FoF1 ATP synthase the membrane protein, and synthesizes
ATP from ADP and Pi when protons pass through Fo the other unit of the ATP synthase. The
structure of F1, consists of 33, is pseudo three-fold symmetric:
a rod-shaped,
asymmetric  subunit is surrounded by an 33 cylinder (1). It was predicted that the
proton-flow through Fo induces the rotation of , forcing the conformational change of three
catalytic  subunits to synthesize ATP (2,3). Isolated F1 is water soluble and hydrolyzes ATP
as a reverse reaction. The rotation of  of isolated F1 was shown by chemical (4) and optical
(5,6) methods. The recent development of single molecule techniques has allowed to observe
the rotation directly under optical microscopes. The 33 cylinder was fixed on a surface, and
the  was labeled with an actin (6,7), a single fluorophore (8) or a bead (9). These work
showed F1 rotates with 120o discrete steps each driven by a single ATP, and the 120o step is
further resolved into 90o and 30o substeps induced by ATP-binding and product release (ADP
and/or phosphate), respectively.
Although several kinds of crystal structures of F1 were solved (1,10,11), it is unknown
how close these are to the structure of the rotating F1, or to which rotation states these crystal
structures correspond. To investigate the structure of rotating F1, we applied a single pair
FRET technique (12,13): we measured FRET between a donor (Cy3) on one of three  and
an acceptor (Cy5) on  of a single thermophillic F1 fixed on a glass surface (14). Because the
yield of FRET strongly depends on the distance between two dyes, would change as  rotates
to move its position relative to 33 cylinder (Fig. 1).
Cy3 was selectively excited with a green laser, and the emission of Cy3 and Cy5 was
simultaneously observed under an epi-fluorescence microscope (15). The time course of the
fluorescence of Cy3 and Cy5 (Fig. 2, A to C) showed alternative and stepwise intensity
changes in the presence of ATP, indicating the repetitive change of the FRET yield (16). The
change in the FRET yield, which presumably represents the rotation of , was faster for higher
[ATP], as expected for the ATP-dependent rotation. When Cy3 lost its acceptor pair due to
Cy5 bleaching, the fluorescence of Cy3 increased (arrow in Fig. 2A). If Cy3 bleached faster,
both disappeared simultaneously (arrows in Fig. 2, B and C). The relationship between Cy3
and Cy5 fluorescence shows two FRET states (Fig. 2, D to F).
The transition rate of the high and low FRET states, defined as averaged and inverted
dwell, (1/<H>,1/<L>), was proportional to [ATP] (Fig. 3), indicating the change in the
FRET yield represents rotation steps. The ratio of dwell of the high and low FRET states,
<H>/<L>, was about two (2.30.3 [avg  s.e., n=15]), suggesting the high and low FRET
states correspond to two and one of three rotation steps. The ATP binding rate, Kon, was
obtained as the slope of the graph for the low FRET state, and twice the slope for the high
FRET state as (10.91.3) and (9.81.4)106M-1s-1, respectively. These values are somewhat
smaller than those obtained by previous single molecule work, 15-30106M-1s-1 (7,8,9) and
obtained from the rate of ATP hydrolysis of non-labeled F1, (16.50.2)107 M-1s-1 (Fig. 3),
presumably due to labeling or fixation to the surface. The rate of the rotation, which obtained
as 1/(<H>+<L>), was also proportional to [ATP] (Fig. 3).
To clarify whether the high and low FRET states include one and two steps, we measured
the histogram of the dwell  multiplied by [ATP] (Fig. 4). If a state consisted of one or two
steps, the histogram of [ATP] would follow equations exp(-Kon[ATP]/) (solid lines in Fig.
4) or exp(-Kon[ATP]/) (dashed lines in Fig. 4), respectively. Whereas the histogram of low
FRET state fits to both the distributions, that of the high FRET state fits better to the two-step
distribution than the other. Fitting one- and two-step distributions to the histograms of the low
and high FRET states gave consistent Kon of 6-8106 M-1s-1, values close to those obtained by
slope of ATP-vs-rate graph (10106M-1s-1). Thus, the distribution of [ATP] is consistent
with the model in which the high and low FRET states consist of two and one steps.
The FRET yield f of the high and low FRET states was calculated as 0.560.06 and
0.150.03 (17), values corresponded to (5.70.7) and (7.90.8) nm of the distance between
two dyes, respectively (18). Because the high FRET state consisted of two rotation steps, the
distance between two dyes would change as 5.7–5.7–7.9 nm.
Three kinds of crystal structures of MF1 were solved: a “native MF1” structure in which
AMP-PNP and ADP occupy its catalytic sites (1), an “MF1-DCCP” structure which is
inhibited with dicyclohexylcarbodiimide(DCCP) and has the same nucleotides with the native
MF1 (10), and an “(ADP AlF4-)2MF1” structure which is inhibited with alminium-fluoride and
has two ADP AlF4- and one ADP (11). A triangle made of three 26 was used as references of
the positions of amino acids, and a line perpendicular to the triangle was assumed to be a
rotation axis (19). The conformation of  inside the cylinder is consistent among these crystal
structures, whereas the protruding portion of  (top part in Fig. 1A) in native MF1 is twisted
clockwisely by 1-20o compared with that in the other MF1 structures (11). The protruding
portion in Eschericia coli F1 ( only) (20) and thermophillic F1 (Y. Shirakihara et al,
unpublished structure) is twisted further. The crystal packaging might affect the protruding
portion. The position of the residue labled with the acceptor, which is on the protruding
portion of , is variable wheras that with the donor is more consistent among these structures
(Fig. 1, B and C).
We calculated the acceptor position from three donor positions and three distances (red
dots in Fig. 1, B to D) (21). While the measured acceptor position somewhat deviated
downwards from the MF1 structures (red dots, Fig. 1B), was consistent in the rotation angle
(Fig. 1C). Note that the angle had to be at the middle of two donor sites, because the FRET
yield of two rotation states were the same.
Two rotation states separated by 30o have been known (9). At low [ATP], F1 spends most
of time waiting for ATP binding (ATP-waiting state). The ATP binding induces a 90o-substep
counter-clockwisely to reach an intermediate state, followed by a 30o substep induced by the
product release. Because the lifetime of the ATP-waiting state (10-1-101 s) is 102-104 times as
long as that of the intermediate state (10-3 s) (9) at low [ATP] we used, the conformation
obtained here should correspond to the ATP-waiting state. Thus, the consistency of our FRET
result and the MF1 structures suggests the MF1 structures are close to the ATP-waiting state.
On the other hand, the measured rotation angle is inconsistent with the crystal structures of
which  subunits rotate by 30o counter-clockwisely (Fig.
1D), suggesting the crystal
structures do not represent the intermediate state.
Two rotation models, tri-site and bi-site mechanisms, have been proposed (22). In the
tri-site mechanism, F1 has two and three nucleotides in the ATP-waiting and the intermediate
states, whereas in the bi-site mechanism, one and two nucleotides in these states. Because the
crystal structures have two or three nucleotides, the consistency of the ATP waiting structure
with the crystal structures suggests F1 on ATP-waiting state has two nucleotides and waiting
for the third nucleotide. Thus, the tri-site model would fit better to our results. The rotation
angle of  of the native and (ADP.AlF4-)2MF1 structures show difference consistent with the
tri-site model in the diection, although the difference is smaller than the full 30o substep (11).
Recent biochemical assay also suggests tri-site mechanism (22).
The mutation to one of three  might make the binding of ATP slower than the other ,
and the high and low FRET states might consist of one and two rotation steps. If this were the
case, the measured acceptor position (yellow dots, Fig. 1 B to D) would be closer to the MF1
structures of which  subunits rotate by 30o, and the bi-site mechanism would be more
appropriate. However, this is less likely because the shape of the dwell distribution of the high
FRET state fits to two-step distribution.
We demonstrated the single pair FRET technique is useful to investigate the structure of a
single protein at work. This technique will also be applicable to proteins on a membrane such
as a whole ATP synthase, because it does not depend on firm fixation of proteins on a surface
unlike other single molecule techniques.
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14. Cy5-maleimide was prepared by mixing 4.1 mM Cy5-OSu (Amersham Pharmacia) and
3.5 mM N-[2-(1-Piperazinyl)ethyl]maleimide, dihydrochloride (Dojin, Tokyo) in
dimethilfolmamide at 40oC over night (23). The reaction was terminated with 20 mM
glycin.
A cysteine of a mutant subcomplex of F1 ((C193S)3(His10-tag at
N-terminus)3(S107C)) derived from thermophillic Bacillus PS3 was labeled with
Cy5-maleimide by mixing the mutant F1 with two molar excess of Cy5-maleimide for 30
minutes at 23oC in buffer A (20 mM 3-[N-morpholino]propane-sulfonic acid-KOH
[MOPS-KOH, pH 7.0] and 100 mM KCl) plus 5mM glycin. A cysteine of a mutant
(S205C) was labeled with Cy3-maleimide (Amersham Pharmacia) by mixing the mutant
 with two molar excess of Cy3-maleimide for 30 minutes at 23oC in buffer A. Free Cy3
and Cy5 were removed on a PD10-column (Amersham Pharmacia). A fraction of 
subunits of Cy5-F1 were exchanged with Cy3- by mixing Cy5-F1 with 10 molar excess of
Cy3- at 45oC for two days. Free  subunits were removed on a Superdex 200 column
(Amersham Pharmacia). The labeling ratio was measured to be 0.7-1 per mol Cy5 and
0.05-0.2 mol Cy3 per F1, using 555Cy3=150,000 M-1cm-1, 555Cy5=15,000 M-1cm-1,
655Cy5=250,000 M-1cm-1, 280Cy3=15,000 M-1cm-1, 280Cy5=25,000 M-1cm-1, 280F1=154,000
M-1cm-1. We diluted the complex to 0.05–0.5 nM into a buffer containing 50 mM KCl, 2
mM MgCl2, 10 mM MOPS-KOH (pH7.0), 0.06–1 M ATP, 70 mM 2-mercaptethanol,
200 g/ml glucose oxidase, 20 g/ml catalase, and 4.5 mg/ml glucose, and sandwiched in
two quarts coverslips to observe under an microscope.
15. A laser beam (532nm, DPSS 532-200, Coherent) was introduced into an objective
(PlanApo x60, NA1.2, water immersion, Olympus) to illuminate fluorophores (8,24). The
emission was collected with the same objective, divided by a dichroic mirror at a wave
length of 620nm, and focused onto a single intensified (VS4-1845, Videoscope)
charge-coupled device (CCD-300T-IFG, Dage-MTI) (25). Bandpass filters (Chroma) were
used to reduce the cross-talk between Cy3 and Cy5 fluorescence to 3 %. The excitation
efficiency of Cy5 was 1/30 times as small as that of Cy3. Images were recording on a Hi8
video recorder (EVO-9650, Sony), captured with a frame grabber (LG-3, Scion) and
analyzed off-line on a personal computer.
16. Most of FRET pairs (>99 %) were inactive, showing no time-dependent change of the
yield of FRET. Single molecule studies on the F1 rotation consistently show a few fraction
of F1 to be active on a surface, presumably due to the surface denaturation (7,8,9).
17. The FRET yield f was obtained as f=1-Cy3/Cy3noFRET=Cy5/(Cy3+Cy5), where
Cy3noFRET is the intensity of Cy3 without FRET and  is the ratio of the intensity change
of two dyes, Cy3/Cy5 (Fig. 2E).
18. The distance between the donor and the acceptor was calculated as R=Ro(1/f-1)1/6 where f
is
the
FRET
efficiency
and
Ro
is
the
förester
distance
determined
as
Ro=[(8.791017)n-4k2QDJ]1/6 where J, the overlap integral, was measured to be
8.8510-13M-1cm3, QD=0.25 is the quantum yield of the donor (26), n=1.33 is the
refractive index, and 2=0.6670.420 is the orientation factor (27). Error of Ro was
estimated as Ro(2)/(62). Resulting Ro was 5.90.6 nm. Error of R was estimated as
(Ro6f2/R5)f+(Ro/R)Ro where f is standard error of f.
19. H. Wang, G. Oster, Nature 396, 279 (1998).
20. A. C. Hausrath, G. Gruber, B. W. Matthews, R. A. Capaldi, Proc. Natl. Acad. Sci.
U.S.A. 96, 13697 (1999).
21. The possible positions of the acceptor were calculated using Monte-Carlo simulation :
we repeatedly and randomly generated one of possible positions of donor (28) and three
distances within the error to determine a position of acceptor.
22. J. Weber, A. E. Senior, J. Biol. Chem. 276, 35422 (2001).
23. T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, T. Yanagida, Nature 374, 555 (1995).
24. I. Sase, H. Miyata, S. Ishiwata, K. Kinosita, Jr, Proc. Natl. Acad. Sci. U.S.A. 94, 5646
(1997).
25. K. Kinosita, Jr, et al., J. Cell Biol. 115, 67 (1991).
26. The quantum yield of dyes was measured to be 0.25 for Cy3 on F1, 0.03 for Cy3 in water,
0.26 for Cy5 on F1 and 0.25 for Cy5 in water using the quantum yield of Rhodamine-B in
ethanol (=0.49) as a standard (29).
27. The orientation factor  is defined as 2=<(cosTcosAcosD)2>corn, where T is the angle
between the donor emission and the acceptor absorption transition moment, A and D are
the angles between donor-acceptor connection line and the donor emission and the
acceptor absorption transition moment, respectively, and <>corn is average over the
fluctuation cone of the donor and the acceptor. Average and standard deviation of 2 over
possible T,A,D were calculated. The cone angle of nano-second fluctuation (X where
X=A or D) of the dyes was estimated from an equation rinf=(ro/2)cosX(1+cosX), where
rinf is the anisotropy at infinite time, which was calculated from an equation
r=(ro-rinf)/(1+3/r)+rinf, in which r is the steady state anisotropy, ro is the limited
anisotropy,  is the fluorescence lifetime of the dye, and r is the rotation relaxation time of
the dye. Lower limit of ro was measured as the steady state anisotropy of the dyes in
glycerol at 0oC to be 0.36 for Cy3 and 0.39 for Cy5. /r was estimated as
(/r)solutionQprotein/Qwater, where Qprotein and Qwater is the quantum yield of the dye on protein
and in water (26), and (/r)water is /r in water which was measured as
(/r)water=(ro/rwater-1)/3 where rwater is the anisotropy of dye in water. The steady state
anisotropy, which was measured to be 0.24 and 0.17 in water and 0.29 and 0.30 on the
proteins for Cy3 and Cy5, gave D>34o and A>45o for Cy3 and Cy5, respectively.
28. The amino acid labeled with the acceptor (107 in thermophillic F1 corresponds to 99 in
MF1) is not seen in the crystals. The possible positions of the residue of 99 were
calculated from the closest amino acids available on both sides (96 and 101). For the
native MF1, missing 91-96 and 101 were compensated using MF1-DCCP structure
before the calculation.
Possible positions of the donor and the acceptor dyes were
obtained as the region reachable with the linkers of dyes (1.5 nm for Cy5 and 0.9 nm for
Cy3) from the possible position of the residues labeled.
29. M. Ishikawa, K. Hirano, T. Hayakawa, H. Shigeru, S. Brenner, Jpn. J. Appl. Phys. 33,
1571 (1994).
30. H. Noji, et al., J. Biol. Chem. 276, 25480 (2001).
f1ref
1. We thank T.Nishizaka, T.Ariga, Y.Shirakihara for critical discussions. This work was
supported by JSPS (RY), Grants-in-Aid from the Ministry of Education, Science, Sports,
Culture of Japan and Keio University Special Grant-in-Aid.
Fig. 1. Visualization of the rotation of F1 with the single pair FRET. (A) The principle of
the experiment. (B to D) Structure of (ADP.AlF4-)2 MF1 (11) with possible positions of
FRET pairs in the native MF1, (ADP.AlF4-)2MF1, and MF1-DCCP structures (blue, purple and
black circles) (28). Red and yellow dots are our results (21). (B) Side view. The putative
rotation axis is shown. (C) Top view. (D) Same as C except  of the crystal structures are
rotated counter-clockwisely by 30o around the putative rotation axis .
Fig. 2. Change in the FRET yield during the rotation. (A to C) The time course of the
fluorescence intensity of Cy3 and Cy5 at various [ATP]. (D to F) Relationship between Cy3
and Cy5 fluorescence corresponds to graphs A to C, regions from the left edges to the arrows.
Lines represent the backgrounds measured as the nearby fluorescence for Cy3 (red) and Cy5
(black) channels.
Fig. 3. Dependence of the rate of the high and low FRET states and the rotation on [ATP].
For comparison, the rate of ATPase of F1 without the subunit exchange ((C193S)3(His10-tag
at N-terminus)3(S107C)) is shown (30). Lines show linear fits with slopes of (4.90.7),
(10.91.3), (3.30.4), and (16.50.2) 106 M-1s-1 for the high and low FRET states, the
rotation, and ATPase (< 2 M), respectively.
Fig. 4. Histogram of the dwell time of the high (black) and low (orange) FRET states. Solid
curves show fits to the single-state distribution, Constantexp(-Kon[ATP]) where
Kon=(6.00.4)106 M-1s-1 (red, 2=24) and (3.20.6)106M-1s-1 (black, 2=142) for the high
and low FRET states, respectively. Dashed curves represent fits to the two-state distribution,
Constantexp(-Kon[ATP])
where
Kon=(13.40.5)106
M-1s-1
(2=28)
and
(7.80.6)106M-1s-1 (2=76) for the high and low FRET states, respectively. The region
0–0.033 s M is missing because is below the temporal resolution (0.033s) at 1 M ATP.