The EMBO Journal (2006) 25, 4567–4576 | & 2006 European Molecular Biology Organization | All Rights Reserved 0261-4189/06
www.embojournal.org

THE

EMBO
JOURNAL

Fast-scanning atomic force microscopy reveals
the ATP/ADP-dependent conformationalchanges of GroEL

Masatoshi Yokokawa*,1, Chieko Wada1,
Toshio Ando2, Nobuaki Sakai3, Akira Yagi3,
Shige H Yoshimura1 and Kunio Takeyasu1
1
Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto
University Graduate School of Biostudies, Kitashirakawa Oiwake-cho,
Sakyo-ku, Kyoto, Japan, 2Department of Physics, Faculty of Science,
Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, Japanand 3OLYMPUS Corporation, Hachioji, Tokyo, Japan

In order to fold non-native proteins, chaperonin GroEL
undergoes numerous conformational changes and
GroES binding in the ATP-dependent reaction cycle. We
constructed the real-time three-dimensional-observation
system at high resolution using a newly developed
fast-scanning atomic force microscope. Using this system,
we visualized the GroES binding to and dissociationfrom individual GroEL with a lifetime of 6 s (k ¼ 0.17 s1).
We also caught ATP/ADP-induced open–closed conformational changes of individual GroEL in the absence ofqGroES and substrate proteins. Namely, the ATP/ADPbound GroEL can change its conformation ‘from closed
to open’ without additional ATP hydrolysis. Furthermore,
the lifetime of open conformation in the presence of ADP(B1.0 s) was apparently lower than those of ATP and
ATP-analogs (2–3 s), meaning that ADP-bound open-form
is structurally less stable than ATP-bound open-form.
These results indicate that GroEL has at least two distinct
open-conformations in the presence of nucleotide;ATP-bound prehydrolysis open-form and ADP-boundopen-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the ‘from open
to closed’ conformational change of GroEL.
The EMBO Journal (2006) 25, 4567–4576. doi:10.1038/
sj.emboj.7601326; Published online 14 September 2006
Subject Categories: proteins; structural biology
Keywords: atomic force microscopy; chaperonin;
two-dimensional protein crystal

Introduction
The atomic force microscopy (AFM) (Binnig et al, 1986;
Hansma et al, 1988) is increasingly being used for studying
biomolecules at the single molecule level. In particular, theAFM has been used to measure both structural and mechan*Corresponding author. Laboratory of Plasma Membrane and Nuclear
Signaling, Kyoto University Graduate School of Biostudies,
Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan.
Tel./Fax: þ 81 75 753 6852; E-mail: yokokawa@lif.kyoto-u.ac.jp
Received: 21 February 2006; accepted: 14 August 2006; published
online: 14 September 2006
& 2006 European Molecular Biology Organization

ical properties of individual DNA and proteins in physiologically relevant buffers (Oberhauser et al, 1999; Allison et al,
2002; Fotiadis et al, 2002; Sekiguchi et al, 2003; Yang et al,2003). However, such investigations have been restricted by
the inherent slow scan speed of AFM necessary for the nanoscale resolution. The scanning rates of commercially available AFMs are usually several seconds to minutes per frame,
and therefore insufficient to monitor many physiologicalprocesses in cells that occur at much faster rates. Recently,
this limitation has been challenged by using fast-scanning
AFM (Viani et al, 2000; Ando et al, 2001), which can capture
images in the subseconds order. In this work, we used a
newly developed fast-scanning AFM to observe the behavior
of individual GroEL at the single molecule level, and demonstrated the ability of fast-scanning AFM to analyze themillisecond-order reaction at nanometer resolution.
Many newly synthesized proteins and denatured proteinsin cells must undergo a series of folding processes to achieve
their proper three-dimensional (3D) structures to become
functionally active (Hightower, 1991; Bukau and Horwich,
1998; Hartl and Hayer-Hartl, 2002). The efficient folding ofmost proteins requires the assistance from molecular chaperones that prevent protein misfolding and aggregation in thecrowded environment in the cells. One of the best-characterized chaperone systems is the Escherichia coli chaperonin
GroEL–GroES system (Bukau and Horwich, 1998; Ranson
et al, 1998; Grallert and Buchner, 2001). The detailed 3D
structures of GroEL, GroES and the GroEL/ADP/GroES
complex were resolved by X-ray crystallography (Braiget al, 1994; Hunt et al, 1996; Mande et al, 1996; Xu et al,
1997; Chaudhry et al, 2003) and electron microscopy(Chen et al, 1994; Schmidt et al, 1994; Ranson et al, 2006)(Figure 1). GroEL consists of two homo-heptameric rings thatare stacked back-to-back, whereas GroES is a single homoheptameric ring. The chaperonin reaction cycle begins withthe binding of a polypeptide (unfolded protein) and GroES to
one side of GroEL, which results in a formation of the GroEL/
polypeptide/GroES complex in the presence of nucleotides.
Once the GroEL/polypeptide/GroES complex is formed, thepolypeptide chain is refolded toward its functional conformation in a large central cavity in the GroEL ring (in cisconformation). Then, the GroEL/GroES complex decays witha lifetime of 8–15 s by the dissociation of GroES, followed bya release of the trapped protein from the cavity (Burston et al,
1995; Weissman et al, 1996; Rye et al, 1997, 1999). This final
step, GroES dissociation, was triggered by the binding of ATP
to the opposite ring (in trans-conformation) (Burston et al,
1995; Hayer-Hartl et al, 1995; Rye et al, 1997).
It is clear that ATP and GroES bindings play essential roles
in chaperonin functions, but the details and the mechanismsof the ATP-dependent reaction cycle of GroEL–GroES system
are still unclear although a number of reaction models, suchas a two-timer model (Taguchi et al, 2001; Ueno et al, 2004)
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GroEL/ES mechanism revealed by fast-scanning AFM
M Yokokawa et al

Figure 1 3D structure of GroEL, GroEL–ATP complex and GroEL–
GroES–ADP complex. (A) The crystal structure of GroEL (Braiget al, 1994); pdb 1GRL. (B) The cryo-EM map of GroEL(D398A)–
ATP complex (Ranson et al, 2001); pdb 2C7E. (C) The crystal
structure of GroEL–GroES–ADP complex (Xu et al, 1997); pdb
1AON. The side views (Upper) and the top views (Lower) of Van
der Waals space-filled models highlighting the individual ring
structures of GroEL (lower GroEL ring, red; upper GroEL ring,green) and GroES (yellow), are shown. Scale bar represents
5.0 nm. Figures were generated using RasMol (http://www.umass.
edu/microbio/rasmol/).

and a single-timer model (Rye et al, 1999), have been
proposed. Especially, the structural consequences of ATP/ADP binding to GroEL remain poorly understood. To understand their structure and functional arrangement in 3Ds inphysiological conditions, AFM observation has been useful(Radmacher et al, 1994; Thomson et al, 1996; Viani et al,
2000; Schiener et al, 2005). In this study, we used the prototype fast-scanning AFM, which is 4100 times faster thanconventional AFM, and constructed a real-time, single-molecule observation system to analyze molecular 3D-structuresand dynamics (kinetics) at the same time. This techniqueallows us to visualize and analyze the association anddissociation reaction of individual GroEL–GroES complex in
the presence of ATP, and the ATP/ADP dependent conformational changes of GroEL without GroES at the single molecule
level in real time.

Figure 2 Topographic high-resolution image of GroEL by the fastscanning AFM. The GroEL molecules were lightly absorbed on amica surface in buffer solution. (A) This is an image of a series offast-scanning AFM images. In this image, the central channels of the
GroEL molecules are visible. The image (240 180 nm2, 192 144
pixels) was taken with a scan rate of 2 frames/s. Scale bar
represents 50 nm. (B) In the lower left area of the image, theupper-heptameric ring of GroEL was removed, which had beenscanned with higher tapping force (see Materials and methods fordetails). The height difference between the normal GroEL and thehalf GroEL is about 7.0 nm, in good agreement with the height of asingle ring of GroEL (Braig et al, 1994). Scan size 400  300 nm2,
192 144 pixels. Scale bar represents 50 nm. The Z-scale is 20 nm.
(C) Illustration of sample geometry.

Results
Imaging GroEL in solution
In order to detect the reactions and conformational changes
of individual GroEL at the single molecule level in real time,
the key techniques were the preparation of a flat and stable
2D GroEL crystal layer and the 3D observation by our fastscanning AFM. GroEL was directly immobilized on a mica
surface through electrostatics force without any modificationof the GroEL molecule such as replacement of amino-acidsequence, fluorescent labeling or chemical treatments. This
immobilization force was relatively loose, so that it would
allow movements and flexible conformational changes of
GroEL molecules on the mica surface.
Fast-scanning AFM demonstrated that GroEL was highly
packed and aligned (an end-up orientation), forming a layer
structure on the mica surface (two-dimensional (2D) protein
crystal) in our experimental condition (Figure 2A). In this4568 The EMBO Journal VOL 25 | NO 19 | 2006

figure, the central channel of GroEL ring could be resolved.
The diameters of the GroEL ring and the central channel
measured from these images were 15.471.9 and 4.771.1 nm,
respectively, in good agreement with previous AFM(Mou et al, 1996), EM (Chen et al, 1994) and X-ray crystallography (Braig et al, 1994) data (Figure 1A, top view), whichshow that the outer and inner diameters of GroEL are about
14.0 and 4.5 nm, respectively. Figure 2B shows the mechanically bisected GroEL layer. The height difference between thenormal GroEL and the half GroEL was about 7.0 nm, precisely
the same as a single heptameric ring of GroEL in transbased on X-ray crystallography (Braig et al, 1994). The surface of GroEL layer that is composed by GroEL 14-mers wasbasically flat in the absence of nucleotide. To quantify andcompare the surface roughness in each image, we measured& 2006 European Molecular Biology Organization

GroEL/ES mechanism revealed by fast-scanning AFM
M Yokokawa et al

Figure 3 Single-molecule imaging of chaperonin GroEL dynamics in the presence of ADP. Time-lapse sequential AFM images of GroEL in abuffer solution on mica surface were taken in the presence of 50 mM ADP alone with 192 144 pixels at a rate of 1 frame per second. Scale bars
represent 50 nm. The Z-scale is 7.5 nm. In the AFM images, taller structure can be seen as the bright (white) features. As identified in the open
white circle at 0 s, a 1 nm taller structure of GroEL (open conformation) than surrounding GroEL layer (closed conformation) had turned into
closed conformation. Open–closed conformational changes of GroEL were repeatedly observed. To remove the effect of ATP contamination in
ADP solution, we used hexokinase and reduced ATP concentration less than 0.1 nM in 50 mM ADP solution.

the root mean square (RMS) value of each image. In this case,
RMS value was 0.6070.02 nm (mean7s.d.).
Our fast scanning AFM observation also demonstrated thatwhen the density of the GroEL molecules on a mica surface
is low and does not form a complete 2D protein layer, GroEL
could randomly diffuse on the mica surface with the averaged
2D diffusion coefficient of 2.271.5 nm2/s (mean7s.d.).
Namely, GroEL molecules frequently get out of and come inthe scanning position (line) during the 1D (2D) observation
by AFM (Thomson et al, 1996; Viani et al, 2000). This meansthat the previously demonstrated 1D or 2D observation
techniques, which have much higher time resolution thanthe 3D-observation technique, are not suitable for a long-term(a few seconds) observation. Therefore, in this study, we
utilized both 2D observation for short-time (o1 s) observations and 3D observation for long-term observations(several seconds).
ATP/ADP mediated open–closed transition of GroEL
To investigate the structural influence of nucleotides, a seriesof AFM images in Figure 3 (see Supplementary Movie 1)
are taken in a buffer solution containing 50 mM ADP without
GroES. In this experiment, the ATP contamination in ADP
solution was removed by hexokinase (Motojima and Yoshida,2003) to less than 0.1 nM in 50 mM ADP solution. Under these
conditions, we observed some taller features (white spots inthe figure) on GroEL layer (RMS value is 0.7870.03 nm in thepresence of 50 mM ADP). The most straightforward interpretation of our results is that the height fluctuation corresponds
to the conformational change of GroEL during nucleotide
binding to GroEL. These taller GroELs were 0.970.2 nm taller
than the surrounding GroELs (Figure 4A). This elongation
value is very similar to the height difference between the
cis-ring and trans-ring conformations of GroEL estimatedfrom 3D-model of the GroEL/ADP/GroES complex (Xu et al,1997), and agrees well with cryo-EM studies (Roseman et al,
1996; Ranson et al, 2001). This height difference wasalso observed in the presence of ATP and nonhydrolysable
ATP analogs (ATPgS, AMP-PNP and ADP þAlFx), but notobserved in the absence of nucleotide (data not shown).
Therefore, we concluded that, in the presence of nucleotides,
GroEL molecules had at least two different conformations,
one is open (elevated) conformation and another is closed(compact) conformation.
& 2006 European Molecular Biology Organization

Figure 4 Histograms of the height differences between the top of
the taller structure of GroEL and top of the surrounding GroEL layer.
Statistical section analyses of GroEL were performed using the datain the presence of (A) 100 mM ADP and (B) 2 mM ATP and 25 nM
GroES. The lines are Gaussian fits of the height difference datefor open-conformations of GroEL (solid line) and GroEL–GroES
complex (broken line).

In order to monitor the conformational changes of GroEL
with higher time resolution, we used 2D-observation
method (Viani et al, 2000), in which a single line wasrepeatedly scanned at a rate of 250–1000 Hz. This approachclearly demonstrated that both of the ‘closed to open’ andthe ‘open to closed’ conformational changes were observed
as 1 nm height difference, and completed within at least
10 ms in most GroEL molecules (more than 95%)
(Figure 5). After this rapid ‘closed to open’ conformationalchange, GroEL kept an open conformation for a few
seconds. These properties of the open–closed conformational
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GroEL/ES mechanism revealed by fast-scanning AFM
M Yokokawa et al

Figure 5 Single-line-scan AFM imaging of chaperonin GroEL dynamics. Single-line-scan (2D observation) AFM images of GroEL in
the buffer A on mica surface were taken in the absence (A) andpresence of 50 mM ADP (B). (Scanning rate of 500 Hz, scan scale(Y-axis direction) of 162 nm and Z scale of 10.0 nm) In these AFM
images, individual GroEL molecules can be seen as tube features(Viani et al, 2000). Lower panels indicate the X-axis cross-sections
positioned at the white dotted lines indicated by the arrows in the
AFM images, which represent typical height fluctuations under
the conditions. One nanometer taller structures (open-conformations) of GroEL and height fluctuations can be seen as the bright(white) and long tube features in the presence of ADP (B), but not in
the absence of nucleotide (A). These open–closed conformationalchanges of GroEL were repeatedly observed. Both the ‘closed to
open’ and the ‘open to closed’ conformational changes in this image
were completed within 5–10 ms. After this rapid ‘closed to open’
conformational change, GroEL kept an open conformation for along time.

changes were also observed in the presence of ADP, ATP
and ATP analogs.
We then examined the nucleotide concentration dependency of the GroEL conformational change (Figure 6). The
ratio of the GroEL molecules in the ‘open’ conformation
was counted in the presence of various concentrations(0–1000 mM) of nucleotides, and the data were fitted with
the Hill equation. The Hill constants (nH) and K1/2 values are
summarized in Figure 6A (inset). The K1/2 values of ATP and
ATPgS are very similar (6–8 mM), whereas that of ADP ismuch higher (B80 mM). At high concentrations of nucleotide, no more than 75% of GroEL had the open-form in
our experiments (Figure 6A). Although we used purified
proteins, a trace amount of the contamination of endogenous
substrate protein could be contained and inhibit the conformational changes of some of GroEL molecules (up to 25%) in
our experimental condition. Such decrement in the percentage of the open-form GroEL by contamination of substrate
proteins has been previously demonstrated by biochemical
experiments (Jackson et al, 1993).
The lifetimes of the open conformation (open time)and closed conformation (closed time) were measured in
the time-lapse 3D AFM images. The histograms of both
open- and closed time in the presence of ADP, ATP or ATP
analogs showed single exponential decays (Figure 7), indicating that the ‘open to closed’ and ‘closed to open’ conformational changes of GroEL in the presence of nucleotides werestochastic processes, which did not require ATP hydrolysis.
The histograms were fitted to a single-exponential model to4570 The EMBO Journal VOL 25 | NO 19 | 2006

Figure 6 Occurrence frequency of the open-conformation of GroEL
with increasing ATP/ADP/ATPgS concentration. Plot (A) shows
the per cent occurrence of the open-form GroEL with increasing
concentrations in the range of 0–1000 mM of ATP, ADP or ATPgS.
(B) The same data in the range of 0–50 mM of ATP, ADP or ATPgS.
The solid line in each case shows the optimal fitting curve of thedata to the Hill equation. The parameters K1/2 and Hill constant (nH)are summarized in the inset table in (A).

obtain the rate constant of the nucleotide-induced conformational change; F(t) ¼ C1 k1 exp(k1t), where F(t) is the number of open form (or closed form) that had an open (orclosed) time t, C1 is the number of the total events and k1 is
the rate constant (kopen-closed and kclosed-open). The obtained
rate constants at various concentrations of nucleotides are
summarized in Table I. At the low concentration of nucleotide, the rate constant of the open to closed conformationalchange did not depend on the nucleotide concentration,
whereas that of the closed to open conformational change
varied depending on the concentrations of ADP, ATP or
ATPgS. This result indicates that the % occurrence of the
open-form GroEL is strongly dominated by the rate constantof the ‘closed to open’ conformational change. Therefore, theresult in Figure 6 indicates that the ‘from closed to open’
conformational change is a cooperative process for consumption of nucleotides.
Dynamics of the GroEL–GroES interactions
with ADP/ATP
An addition of GroES to the imaging solution without nucleotide did not cause any apparent changes in the surface feature& 2006 European Molecular Biology Organization

GroEL/ES mechanism revealed by fast-scanning AFM
M Yokokawa et al

Figure 7 Statistical analysis of conformational changes of GroEL in the presence of nucleotides. Histograms in (A) and (C) show the typical
distributions of the lifetime of open-conformation of GroEL in the presence of 50 mM ADP and 5 mM ATP, respectively. Histograms in (B) and (D)show the typical distributions of the lifetime of closed-conformation of GroEL in the presence of 50 mM ADP and 5 mM ATP, respectively. Some
GroELs that do not undergo conformational changes at all over the period of observation were excluded from the analyses. The histograms
were fitted with a single–exponential function as the following equation: F(t) ¼ C1k1 exp(k1t), where F(t) is the number of open-form (orclosed-form) GroEL that had an on (or off) time t, C1 is the number of the total events and k1 is the rate constant. The rate constants (k1) wereobtained by the nonlinear least-squares curve fitting method. The same analyses were performed in the presence of different concentrations of
nucleotides. The obtained fitted curves were summarized in the inset figures. The obtained rate constants in each nucleotide condition are
averaged and summarized in Table I.
Table I Rate constants of ‘open to closed’ and ‘closed to open’ conformational changes of GroEL with increasing nucleotide concentration
Concentration(mM)

kopen-closed(s1, mean7s.d.)kclosed-open(s1, mean7s.d.)Open conformation(%, mean7s.d.)

ADP

10
30
50

1.0570.20
1.1170.25
0.9670.08

o0.1
0.1070.03
0.4270.02

171
771
1873

ATP

1
3
5
7

0.7170.21
0.6870.06
0.5270.06—

o0.1
0.1270.05
0.3770.23
0.6370.16

171
1471
3176
4079

ATPgS

3
5
7

0.4570.12
0.4170.06
0.3070.05

o0.1
o0.1
0.2670.08

170
1173
3074

50
400

0.5270.02
0.3470.02

0.3570.04
0.1270.02

1974
3274

Nucleotide

ADP+AlFx
AMP-PNPThe values of the rate constants are the mean7s.d. of 3–5 independent experiments.

of the GroEL layer (RMS value is 0.5870.03 nm), which isconsistent with the previous experiments (Chandrasekhar
et al, 1986). On the other hand, in the presence of 2 mM
ATP and 500 nM GroES, much taller features than the
open conformation of GroEL were occasionally observed(RMS value of 1.1070.08 nm) (Figure 8A). The histogram& 2006 European Molecular Biology Organizationof the height difference from the surface of the GroEL layer(Figure 4B) has two peaks near 3.970.6 and 1.370.5 nm.
The mean value of the taller one (3.970.6 nm) well corresponds with the height difference between the GroEL/ADP/
GroES complex (Xu et al, 1997) and GroEL (Braig et al, 1994),
and the shorter one (1.370.5 nm) corresponds to the height
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Figure 8 Single-molecule imaging of chaperonin GroEL–GroES dynamics in the presence of nucleotides. Time-lapse sequence AFM images of
GroEL on mica surface in a buffer solution were taken in the presence of (A) 2 mM ATP and 500 nM GroES, (B) 20 mM ADP and 25 nM GroES or(C) 10 mM Caged ATP and 25 nM GroES with 192 144 pixels at a rate of 1 frames pre second. Scale bars represent 100 nm. The Z-scale is 10 nm.
(A) GroES molecules were repeatedly found to bind to and dissociate from GroEL. ATP concentration was kept low (unsaturated condition) inorder to observe and analyze single molecule events. In the AFM images at 0 s, for example, a 4 nm taller structure (GroEL–GroES complex) canbe seen in the right open white circle. (B) In the presence of ADP, extremely stable association of GroES with GroEL (GroEL–ADP–GroES
complex) was achieved. Same results were observed with the wide range of concentration (25–500 nM). This result indicates that tip-sample
interaction in our experiments is basically week enough to keep the GroEL–ADP–GroES complex stable. (C) After UV illumination at time 1 s,
GroES quickly began to associate to and dissociate from individual GroEL with the generation of ATP.

difference between the open- and the closed conformations.
In the series of AFM images shown in Figure 8A (seeSupplementary Movie 2), GroES molecules repeatedly associated with and dissociated from GroEL. Immediately after
the dissociation of GroES, GroEL was found to be ineither open (10% of the total) or closed (90% of the total)
conformation, suggesting that GroEL switches back to theclosed conformation immediately (within 1 s) after the
dissociation from GroES (Figure 9).
The duration time of GroES-unbound form of GroEL in
the presence of a certain concentration of GroES (25 nM) andATP (100 mM) (‘off time’) was measured (Figure 10A). The
histogram of the ‘off-time’ was well fitted to a single exponential curve, indicating that the GroES binding was also
a stochastic process. The association rate constant of GroES
to GroEL (kon) (2.3 107 M1 s1) was calculated by curve
fitting, which is half of the value measured by the previous
bulk-phase experiments (4.6 107 M1 s1) (Rye et al, 1999;
Taguchi et al, 2001; Taniguchi et al, 2004). This is reasonablebecause one side of GroEL ring is inaccessible to GroES in our
experimental condition. Thus, the semiimmobilized GroEL
sample does not lose the activity for GroES binding and the4572 The EMBO Journal VOL 25 | NO 19 | 2006

effect of probe scanning to the GroES binding reaction is low
enough to be ignored.
The duration time of GroES-bound GroEL (‘on time’) in thepresence of 500 nM GroES and 2 mM ATP was measured andare summarized in Figure 10B. This histogram has a maximum peak at 4 s (the average lifetime is 6 s) and does notshow a single exponential decay, indicating that the dissociation reaction of GroES from GroEL is probably not governedby a single rate-determining step, rather obeys two sequential
transitions (two timer model), as previously proposed by
Taguchi et al (2001) and Ueno et al (2004).
½GroEL=GroES ! ½GroEL=GroES !0 ½GroEL þ ½GroES
k2

k2

ð1Þ
The histogram data were well fitted to the equation deduced
from scheme (1): G(t) ¼ C2k2k20 [exp(k2t)exp(k20 t)]/(k20 k2), where G(t) is the number of GroEL/GroES complex
that had a lifetime t, k2 ¼ 0.37 s1, k20 ¼ 0.35 s1 and C2 ¼ 295(the number of the total events). We also analyzed the ATPconcentration dependency on GroES-retained time (Table II).
The time constants of the lifetimes of two independent states& 2006 European Molecular Biology Organization

GroEL/ES mechanism revealed by fast-scanning AFM
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Figure 9 Single-molecule imaging of GroES dissociation fromGroEL in the presence of ATP. (A) Time-lapse sequence AFM imagesof GroEL on mica surface in a buffer solution were taken in thepresence of 2 mM ATP and 500 nM GroES at a rate of 1 frames per
second. Scale bars represent 50 nm. Time 0 was set at the top of thefirst scan. Initially, some GroEL–GroES complex can be seen as
white spots (white arrows, at time 0). Then, the GroES molecules
were dissociated from GroEL during the observation (1–2 s forthe lower complex and 2–3 s for the upper complex). In the caseof the lower one, after GroES dissociation, open conformation wasobserved. (B) Time-lapse sequence cross-sections positioned by theblack arrows (i, ii).

were 3 and 2–3 s, respectively, and the sum was 5–6 s, which
well agrees with the previous value (7–15 s) estimated inbulk-phase kinetics (Burston et al, 1995; Weissman et al,
1996; Rye et al, 1997, 1999; Taguchi et al, 2001).
Caged ATP was employed in order to directly detect theATP-induced GroEL association with GroES. The fast-scanning imaging of GroEL layer was performed in the solution
containing 10 mM caged ATP and GroES. At a certain time
point during the imaging, ultraviolet (UV) light was irradiatedto the specimen and the observation was continued. The
shutter of the UV light remained open for about 5 s, which
was enough to generate 2 mM or more ATP from 10 mM caged
ATP. After the irradiation, GroES quickly began to associate
to and dissociate from GroEL with the ‘on time’ of 6–7 s(Figure 8C) (see Supplementary Movie 3).
In the presence of 20 mM ADP, the extremely stable association of GroEL with GroES (GroEL/ADP/GroES complex)
was observed (Figure 8B). In the presence of ADP, thelifetime of GroEL/ADP/GroES complex was so long that we
hardly observe the GroES dissociation from GroEL within thetime of observation (the average lifetime is more than 1 min),as predicted by the previous biochemical experiments(Todd et al, 1994; Hayer-Hartl et al, 1995; Motojima andYoshida, 2003).

Figure 10 Statistical analyses of GroES binding and dissociation
events of GroEL in the presence of ATP. Histogram in (A) shows the
distribution of the duration time between the GroES dissociationfrom and the next GroES binding to individual GroEL molecule in
the presence of 25 nM GroES and 100 mM ATP. Some GroELs that do
not undergo conformational changes at all over the period of
observation in the presence of ATP was excluded from the analyses.
The histogram was fitted with a single-exponential function as thefollowing equation: H(t) ¼ C3k3 exp(k3t), where H(t) is the number of GroEL that had an off time t, C3 is the number of the total
events and k3 is the rate constant. The rate constant (k3) was
obtained by the nonlinear least-squares curve fitting method. Then,the association rate constant for GroES binding (kon) was calculated. Histogram in (B) shows the distribution of the lifetime of
GroEL–GroES complex. Individual GroES molecules that attached
to and dissociated from GroEL molecules repeatedly were used forthe analyses over the observation time of B10 min. The solid line
was obtained by the least-square fitting to the equation:
C1k2k20 [exp(k2t)exp(k20 t)]/(k2k20 ) derived from the twostep reaction of scheme (1).

Table II Rate constants of GroES dissociation with increasing ATP
concentration
ATP concentration (mM)
2
20
100

k2 (s1)

k20 (s1)

0.37
0.42
0.39

0.35
0.48
0.47

Discussion
A significant feature of our real-time 3D-observation technique using newly developed fast-scanning AFM is its
capability of directly analyzing conformational changes ofa molecule and inter-molecules interactions at the single
molecule level in the real-time scale. Both the ‘real-time’and the ‘nm-scale’ single molecule observations of structurally unstable biomolecules cannot be achieved by othertechniques, although cryo-EM (Chen et al, 1994; Schmidt& 2006 European Molecular Biology Organization

et al, 1994; Roseman et al, 1996; Ranson et al, 2001, 2006)
can take the ‘static’ images at a nm-scale and fluorescentexperiments, such as FRET experiments (Motojima et al,2004) and stopped-flow experiments (Poso et al, 2004;
Taniguchi et al, 2004), can detect the ‘statistical’ structural
information in real-time. Thus, our experimental method
potentially gives us a new insight into the molecular
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GroEL/ES mechanism revealed by fast-scanning AFM
M Yokokawa et al

dynamics of biomolecular reactions involving structuralchanges. In this study, we demonstrated (i) an ATP/ADPdependent open–closed transition of GroEL without GroES,and (ii) an ATP-dependent association–dissociation eventsof the GroEL/GroES complexes.
Nucleotide-induced conformational change of GroEL
The histogram of height differences between the taller
structure (open-form GroEL) and the surrounding GroEL(closed-form GroEL) in the presence of ATP or ADP (but inthe absence of GroES or substrate polypeptides) showed asharp peak at about 1 nm (Figure 4A), indicating that there is
no structural intermediate. The one-line scanning experiment
revealed that the open–closed transition of GroEL in theabsence of GroES or substrate polypeptides is a very fast
process (within 10 ms; Figure 5B), which is consistent withthe results and predictions from the previous fluorescentexperiments (Poso et al, 2004; Taniguchi et al, 2004).
Therefore, these two structural states of GroEL, open-form
and closed-form GroEL, are clearly distinguishable.
When we kept monitoring a given single GroEL moleculein the presence of nucleotide, we observed repeated conformational changes between open and closed states (seeSupplementary Movie 1). As summarized in Table I, theopen time does not depend on the nucleotide concentration.
On the other hand, the ‘closed time’ was always dependent
on the nucleotides concentration, indicating that the ‘closed
to open’ conformational change requires nucleotide binding.
From these results, we can conclude that the ‘closed
conformation’ is a nucleotide unbound form and the ‘open
conformation’ is a nucleotide bound form, and the open–
closed transition of GroEL is induced by the binding andunbinding of nucleotides to GroEL (on/off process). In thispoint, a structural fluctuation of the nucleotide-bound GroEL
is unlikely; that is, once nucleotide binds to the closed-formof GroEL, it could not be structurally mobile and fluctuate
between open and closed conformations without releasing
the nucleotides. This is further supported by the observation
that, in a very low nucleotide concentration (only a few percent of GroEL molecules have open conformation), open–
closed transition did not ‘continuously’ occur on the same
GroEL molecule (see Supplementary Movie 1), but rather
occurred randomly on separate molecules in the GroEL layer.
It is also notable that both ‘open to closed’ and ‘closed to
open’ conformational changes occur in the presence of ADP
or nonhydrolysable ATP analogs (Figures 3 and 6). Especially,
with regard to the nucleotide concentration dependencyof the kclosed-open value, those for ATP and ATPgS are quite
similar (Table I). This means that the nucleotide-bound
GroEL changes its conformation ‘from closed to open,
and vice versa’ without ATP hydrolysis. On the other
hand, the kopen-closed value in the presence of ADP(B1.0 s1) was apparently larger than those of other nucleotides (0.3–0.5 s1) (Table I), meaning that the ADP-boundopen form is structurally less stable than other nucleotidebound forms. These results imply that there might be two
distinct open conformations in the presence of nucleotide:ATP-bound prehydrolysis open-form and ADP-bound openform, which could not be distinguished in the AFM images.
Based on our observations and other previous reports, thereaction steps of nucleotide-induced conformational changeof GroEL can be depicted as in Figure 11. It has been known
4574 The EMBO Journal VOL 25 | NO 19 | 2006

Figure 11 Proposed reaction model of the ATP-dependent open–
closed transition of GroEL. ATP binds to the closed conformation of
GroEL with higher affinity than ADP and immediately induces
conformational change to open state (GroES accepting form).
Then the hydrolysis of ATP occurs and the open GroEL undergoes
another conformational change to ADP-bound open state, which isless stable than ATP-bound open state. Finally, the open to closed
conformational change of GroEL occurs by releasing the nucleotides
from GroEL subunits.

that the hydrolysis of ATP was strongly coupled with thestructural change of GroEL (Murai et al, 1996; Shiseki et al,2001). Our result in which the kopen-closed value in thepresence of ATP was between those in the presence of ADP
and ATP analogs (Table I) also implies that ATP hydrolysis
can occur in open-form GroEL. Thus, in the ATP-induced
conformational change, ATP binds to the closed state of
GroEL and induces conformational change to the openstate. Then the hydrolysis of ATP occurs and the open
GroEL undergoes another conformational change to the
ADP-bound open state, which is less stable than the ATPbound open state and swiftly releases ADP and returns backto the close conformation (Figure 11).
ATP-dependent association–dissociation of the
GroEL–GroES complexes
The open-conformation of GroEL without GroES (Figure 3)
and the cis-conformation of GroEL in the GroEL/ADP/GroES
complex (Xu et al, 1997) have similar structures (1 nm taller
structures). If we consider these two GroEL structures as thesame or similar ones, it is very interesting that the ‘open to
closed’ conformational change of GroEL without GroES(0.63 s1/2 mM ATP and 0.94 s1/50 mM ADP) is apparently
faster than the dissociation of GroES from GroEL (0.17 s1/
2 mM ATP and 0.00 s1/50 mM ADP) in the presence of thesame concentration of ATP and ADP. Namely, the GroES
binding elongates the lifetime of cis (or open)-conformationof GroEL. Furthermore, their time distributions in the presence of ATP/ADP are very different. These results indicatethat the open–closed transition of GroEL is not the ratedetermining steps in the ATP dependent reaction cycle of
the GroEL–GroES system and GroEL has other mechanisms tocontrol the stable association with and the dissociation of
GroES. With regard to the longer lifetime of GroEL/GroES
complex, it can be assumed that the GroES binding strongly
restricts the ‘open to closed’ conformational change of GroEL.
On the other hand, concerning the molecular mechanism of
GroES dissociation from GroEL, one possibility has been
already shown that the further ATP-binding to or ATP-hydrolysis in the counter ring structure of GroEL (Rye et al, 1997,1999) might be the key reaction of GroES dissociation.
The unique aspect of our experimental system resides inthe specimen; one side of the GroEL ring is inaccessible to
GroES because of the GroEL adsorption with an end-up& 2006 European Molecular Biology Organization

GroEL/ES mechanism revealed by fast-scanning AFM
M Yokokawa et alorientation without introducing any mutations. This enabled
us to analyze the GroEL–GroES interaction at only one sideof the GroEL complex without the influence by the GroESbinding to the other side. Nevertheless, our results of nucleotide concentration dependency of GroEL conformationalchange (Figure 6) are still similar to the results previously
measured by the bulk-phase experiments (Jackson et al,
1993; Poso et al, 2004). The measured lifetime of GroEL/
GroES complexes in the presence of ATP also agreed very
well with those obtained in the completely different single
molecule experimental systems, for example, total internalreflection fluorescent (TIRF) microscopy (Taguchi et al, 2001;
Ueno et al, 2004). This similarity of the lifetimes of GroEL/
GroES complexes measured by our method and TIRF methodsuggests that it is not necessary for both rings of GroEL to beaccessible to GroES for the chaperonin function, although
GroES may be able to bind to both ends of GroEL to form asymmetric complex and the symmetric complex may havesome biological function under physiological condition(Sparrer et al, 1997; Grallert and Buchner, 2001).

Materials and methods
Fast-scanning AFM
Our fast-scanning AFM system was developed based on Ando et al(2001) system with some modifications as follow. We used newly
developed piezo scanners, the resonance frequencies of which are
XY 20 kHz and Z 150 kHz. The small cantilevers, made from silicon
nitride using standard micromachining techniques (Kitazawa et al,2003), were 10 mm long, 2 mm wide and 0.1 mm thick. Theirresonance frequencies in water were about 600 kHz, and thespring constants in water measured by the thermal vibration
method (Hutter and Bechhoefer, 1993) were about 0.1–0.2 N/m.
Each cantilever had an electron beam deposited probe made by
Nano tools GmbH (Munchen, Germany). In order to use thesesmall cantilevers, we combined the AFM and an inverted optical
microscope (IX-71, Olympus, Tokyo, Japan), which was used
for the detection of the motion of mounted cantilevers as angularchanges in the reflected light beam focused on the back of cantilever through the microscope optics (Ando et al, 2001). Thesedevelopments allowed us to take an image within 80 ms withtapping mode in buffer solution at room temperature. The scales ofthe piezo scanner were calibrated against a grid of known
dimensions. The error margins of the outputs of our (Z, X–Y)
piezo scanners were typically within 3%. Time scale typicallyvaried by 7 several % because our AFM system uses PC clock toregulate the timing.
Furthermore, our AFM system contained an UV illumination
system. The UV light provided by the mercury lamp (HBO103W/2,
USHIO) was used to photolyze caged ATP after once passed throughthe (U-MWU2) filter. The mechanical shutter shut and opened theUV light, and the UV light was focused on the top of the probe. The
diameter of the UV spot size was around 1 mm2 including the AFM
scanning area.
Materials
All chemicals used in these experiments were reagent grade. Caged
ATP was obtained from Calbiochem (Darmstadt, Germany). ATPgS,AMP-PNP and hexokinase were obtained from Roche (Penzberg,
Germany). E. coli GroEL and GroES were purified as described in
the previous report (Motojima et al, 2000). The purified GroEL
and GroES were stored at 801C. Purified GroEL and GroES
hardly contained contaminated proteins and showed very low
Trp fluorescence, equivalent to less than 0.2 Trp per GroEL
tetradecamer. GroEL and GroES concentration was determined by
Bradford protein assay (Bio-Rad) calibrated by quantitative
amino-acid analysis. Before using, the stock GroEL and GroES
solution were diluted (50 mg/ml for GroEL in 50 mM HEPES–KOH,
100 mM KCl, 5 mM MgCl2 and 1 mM DTT, pH 7.5; 250 mg/ml for
GroES in 50 mM HEPES–KOH, 100 mM KCl, 5 mM MgCl2 and 1 mM
DTT, pH 7.5).
& 2006 European Molecular Biology Organization

Specimen preparation and fast-scanning AFM observation
For the preparation of AFM samples, a 3.0 ml droplet of GroEL
solution was directly applied onto a freshly cleaved mica surface(1.0 mm2). After an incubation for 30 min at room temperature, thesample was gently washed several times with buffer A (25 mM
HEPES–KOH, 100 mM KCl, 5 mM MgCl2, 10 mM DTT and 25 mM
glucose, pH 7.5) to remove un-adsorbed GroEL and kept in thesame buffer solution until used. Fast-scanning AFM imaging intapping mode was performed in the same buffer solution withor without ATP, ADP, caged ATP, ATP analogs (ATPgS, AMP-PNP
and ADP þAlFx) and GroES (final concentration of GroES was
25 or 500 nM). In the case of using ADP þAlFx, fast-scanning
AFM imaging was performed in the buffer A containing
various concentrations of ADP, 30 mM NaF and 5 mM Al(NO3)3.
The ATP contamination in commercially available ADP, AMP-PNP
or ATPgS was fully hydrolyzed by adding hexokinase for 5 min(final concentration is 0.4 U/ml) to the buffer solution. ATPconcentration in solution was measured with the firefly luciferinluciferase method with a C-100N luminometer (KIKKOMAN,Tokyo, Japan).
All AFM images were obtained at room temperature with ascanning speed of typically 1–4 frames per second for 3Dobservation and 250 Hz (lines per second) for 2D-observation.
The cantilever was driven at the frequency of 590–610 kHz and withthe drive amplitudes of 0.5–2.0 V. The free cantilever amplitude inliquid was set to 1.2–2.0 nm as determined from the force curve. For
fast-scanning AFM observation, the force applied by the cantileverto sample was minimized by setting to the highest possible
amplitude set-point voltage, which was typically above 80% of
free oscillation just close to sample surface. For mechanicallybisecting GroEL molecules, amplitude set-point was set to under
60% of the free oscillation.
In the case of using caged ATP, buffer and sample surface in the
AFM liquid cell was directly illuminated with UV light for 5 s during
fast-scanning AFM observation. Approximately 50% of caged ATP
released ATP under our experimental condition.
Analysis of fast-scanning AFM data
To quantify the molecular motion, we calculated the 2D diffusion
coefficient of a molecule (D2D). D2D was calculated using thefollowing equation:
D2D ¼

2
hDr2D
i
4Dt

ð2Þ

where /Dr22DS is the 2D mean-square displacement of a moleculeduring the time interval Dt. The values of D2D determined fromindividual 50 GroEL molecules were then averaged.
To quantify and compare the surface roughness in each image,
we measured the RMS value of Z-displacements in each image. For
this analysis, we prepared highly occupied GroEL layer, meaning
the same protein density (12774 molecules/162  216 nm2), andmeasured the surface roughness of the top of the extremely flat
GroEL layer. The RMS values of Z-scale determined from individual
40 images were then averaged.
Movie (images) analysis was performed with the ImageSMX(http://www.liv.ac.uk/~sdb/ImageSXM/).
Supplementary data
Supplementary data are available at The EMBO Journal Online(http://www.embojournal.org).

Acknowledgements
We thank Dr F Motojima and Dr M Yoshida for supplying thepurified GroEL and GroES used in our experiments; Dr H Taguchi
and Dr T Mizobata for comments on the manuscripts; Dr H
Sugiyama for the use of a fluorometer. We also thank members of
OLYMPUS Corporation and of our laboratory for helpful discussion
and much technical advice. This work was supported by the Special
Co-ordination Funds, the COE Research Grant and the Basic
Research Grant (B) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and the Measurement
and intellectual infrastructure improvement project for small andmedium enterprise from the Ministry of Economy, Trade andIndustry of Japan.
The EMBO Journal

VOL 25 | NO 19 | 2006 4575

GroEL/ES mechanism revealed by fast-scanning AFM
M Yokokawa et al

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