Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

rstb.royalsocietypublishing.org

Substrate protein dependence of GroEL –GroES interaction cycle revealed by highspeed atomic force microscopy imaging
Daisuke Noshiro1,2 and Toshio Ando1,2Research
Cite this article: Noshiro D, Ando T. 2018
Substrate protein dependence of GroEL – GroES
interaction cycle revealed by high-speed
atomic force microscopy imaging. Phil.
Trans. R. Soc. B 373: 20170180.
http://dx.doi.org/10.1098/rstb.2017.0180
Accepted: 2 March 2018
One contribution of 17 to a discussion meeting
issue ‘Allostery and molecular machines’.
Subject Areas:
biophysics
Keywords:
chaperonin, GroEL, allostery, imaging,high-speed atomic force microscopy
Author for correspondence:
Toshio Andoe-mail: tando@staff.kanazawa-u.ac.jp

Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.
figshare.c.4054898.

1
2

Nano Life Science Institute (WPI NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
CREST, Japan Science and Technology Agency, Tokyo 102-0076, JapanDN, 0000-0001-7024-2054; TA, 0000-0001-8819-154X
A double-ring-shaped tetradecameric GroEL complex assists proper protein
folding in cooperation with the cochaperonin GroES. The dynamic GroEL–
GroES interaction reflects the allosteric intra- and inter-ring communications
and the chaperonin reaction. Therefore, revealing this dynamic interaction isessential to understanding the allosteric communications and the operation
mechanism of GroEL. Nevertheless, how this interaction proceeds in thechaperonin cycle has long been controversial. Here, we directly image thedynamic GroEL–GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, theimaging results obtained under these conditions and our previous results inthe presence of unfoldable substrate are compared. The molecular movies
reveal that the entire reaction pathway is highly complicated but basically
identical irrespective of the substrate condition. A prominent (but moderate)
difference is in the population distribution of intermediate species: symmetric
GroEL : GroES2 and asymmetric GroEL : GroES1 complexes, and GroES–
unbound GroEL. This difference is mainly attributed to the longer lifetimeof GroEL : GroES1 complexes in the presence of foldable substrate. Moreover,the inter-ring communication, which is the basis for the alternating action of
the two rings, occurs at two distinct (GroES association and dissociation)
steps in the main reaction pathway, irrespective of the substrate condition.
This article is part of a discussion meeting issue ‘Allostery and molecularmachines’.

1. Introduction
In bacteria, GroEL assists proper folding of many proteins in cooperation with
its cochaperonin GroES [1,2]. GroEL is a cylindrical protein complex formed by
two heptameric rings stacked back to back, each consisting of identical ATPasesubunits [3]. GroES is a single homo-heptameric ring [4] and binds to the endsof the GroEL cylinder depending on the nucleotide state of GroEL. These
multiple factors involved in the reaction cycle of this system make it difficult
to analyse the reaction cycle with ensemble averaging methods. Nevertheless,several issues concerning this reaction cycle have been revealed by extensive
biochemical and structural studies [5– 7]. Unfolded substrate protein (SP)with exposed hydrophobic residues binds to GroEL at its apical domain [8].
Then, the SP is encapsulated into the hydrophilic cavity of GroEL after its binding to ATP and GroES [9,10]. The encapsulated SP can fold in this environment.
Subsequently, GroES dissociates and then the SP is released into solution.
Because the two rings of GroEL are identical, these processes proceed at eachring. Then, an important question is raised: whether or not the molecular processes
at one ring proceed independently of those at the opposite ring. Biochemical kinetic studies of ATP binding and hydrolysis put forward allosteric regulation withintra-ring positive cooperativity and inter-ring negative cooperativity as regard to
ATP binding [11–13]. Based on this allosteric regulation, a model has been postulated in which only one ring binds GroES throughout the cycle, so that asymmetric
GroEL : GroES1 complexes (referred to as the bullet complexes) are exclusively& 2018 The Author(s) Published by the Royal Society. All rights reserved.

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

complexes, and the bullet complexes formed at the exit of theside pathway can enter either pathway at the respective constant probabilities. In the present HS-AFM study, we image
the GroEL–GroES interaction in the absence and presence offoldable SP and compare the reaction schemes obtained
under the three substrate conditions.

2. Intermediates and reaction patterns

3. Decay kinetics of bullet and footballcomplexes
All histograms of lifetime for bullet complexes (figure 3a,c,e,g;
electronic supplementary material, figures S1 and S2) were

Phil. Trans. R. Soc. B 373: 20170180

To visualize the dynamic GroEL–GroES interaction by
HS-AFM, the D490C GroEL mutant biotinylated at Cys490
with a bifunctional reagent (linker length, approx. 2.5 nm) was
immobilized in a side-on orientation on the two-dimensional(2D) crystal surface of a tamavidin 2 mutein with a lowered
isoelectric point (tamavidin 2-LPI) [42] formed directly on abare mica surface (figure 1a). All imaging experiments were
performed at 4.35 frames s21 (0.23 s per frame) under the conditions of 258C, 100 mM KCl, 25 mM HEPES-KOH (pH 7.4),
5 mM MgCl2, 2 mM ATP, 1 mM GroES, with or without
200 nM of a double mutant of maltose binding protein (DMMBP) denatured with guanidine hydrochloride. Rhodanese
was also used as a foldable SP. In the successive AFM images,
three intermediates appeared (figure 1b; supplementary
movies S1–S3): GroES–unbound, and bullet and football complexes (we express these intermediates as ‘U’, ‘B’ and ‘F’,
respectively). The population ratios of these intermediates are
summarized in table 1, including those obtained in the presenceof a-LA [41] and rhodanese. The population ratio in the absenceof SP was similar to that observed in the presence of a-LA (football complexes were a major intermediate: 53–67%), whereas
bullet complexes most frequently appeared in the presence of
DM-MBP or rhodanese (62–65%) and football complexes
appeared at approximately 30%. A prominent feature observedin the presence of foldable SP was moderately frequent appearance of GroES–unbound GroEL (5–8%), which appeared only
at 0.4% in the presence of a-LA and at 1.3% in the absence of SP.
Next, we analysed the order of association and dissociationof GroES at the two rings of GroEL by choosing the bullet complexes as an initial state (figure 2). As observed previously inthe presence of a-LA [41], these dynamic events are largely
classified into Type I and Type II; in Type I, the cis/transstates interchange between the two rings after a round of dissociation and association of GroES, resulting in polarity
change between the initial and second bullet complexes (i.e.
B ! X ! B; the vertical arrows indicate the polarity ofbullet complexes and ‘X’ denotes F, U or none), whereas in
Type II no cis/trans interchange occurs, resulting in no polarity
change (i.e. B ! X ! B; ‘X’ denotes F or U). Reaction patterns not belonging to either Type I or Type II (i.e. B ! U !
F and B ! F ! U) rarely appeared in the presence of DMMBP (1.8% in total). We omit these cases from the analyses
described below. The population ratios of all reaction patterns
are summarized in table 2. As noted in table 2, the appearance
frequency ratio between Type I and Type II processes is nearly
independent of the substrate condition (Type I : Type II  3 : 1),
although the Type II process appears more frequently in thepresence of a-LA (Type I : Type II  2 : 1).

2

rstb.royalsocietypublishing.org

formed [14]. The GroES-free ring (trans-ring) can bind ATP only
after ATP is hydrolysed in the GroES-bound ring (cis-ring).
Actual ATP binding to the trans-ring induces release of
GroES, ADP and the encapsulated SP protein from the opposite
ring, while the second GroES binds to the trans-ring to form a
new cis-ring [15,16]. Thus, the two rings of GroEL alternatelybind and release GroES and hence alternately function. Nonetheless, symmetric GroEL : (GroES)2 complexes (referred to as
the football complexes) have also been observed under electron
microscopy [17–22]. Moreover, biochemical measurements inthe presence of various concentrations of ATP and GroES
have shown the folding activity to be nearly proportional tothe population of football complexes as well as theabolishment of inter-ring negative cooperativity of ATP bindingat [ATP] greater than approximately 50 mM [21,22]. The structural basis of this abolishment of inter-ring negative
cooperativity has recently been revealed by the atomic structureof football complexes formed by a GroEL mutant with a very
low ATPase activity [23]. In spite of these observations (formore details, see [24]), this model postulates that football complexes appear only as a transient intermediate in the reaction
cycle, at the time when GroES binds to the trans-ring justbefore the completion of GroES release from the cis-ring.
To inspect this model more directly, solution kinetics studies
with fluorescence energy transfer-based GroEL–GroES association detection [25–31] and single-molecule fluorescencemicroscopy observations [32–35] have been performed in thepast two decades. Most of these studies revealed a part of thereaction pathway and detected football complexes not as abriefly subsisting intermediate but as a major one appearing
during the reaction cycle in the presence of unfoldable orfoldable SP. Very recently, dual-colour fluorescence crosscorrelation spectroscopy analysis was performed using
differently labelled GroES [36]. This study showed that footballcomplexes were to an appreciable extent formed only in the presence of unfoldable SP, whereas both in the presence of foldable
SP and in the absence of SP, football complexes were onlytransiently and hence rarely formed. Thus, even with singlemolecule fluorescence approaches, inconsistent results haveemerged. This may be partly due to different dyes [36] anddye-labelling sites being used. More seriously, the two rings of
GroEL are too closely positioned to be resolved with fluorescence methods. Therefore, it is often very difficult to know
from which ring a detected fluorescence signal comes. To
solve this controversial issue definitely, we need a further
direct method to monitor the dynamic binding and release of
GroES occurring in nano-scale space.
Here we used high-speed atomic force microscopy (HSAFM) [37,38] to directly visualize the dynamic GroEL–GroES
interaction with time resolution of 0.23 s. HS-AFM has recently
been used with great success to visualize protein molecules indynamic action without disturbing their function [39,40].
In our previous HS-AFM study [41], we imaged the GroEL–
GroES interaction in the presence of unfoldable SP, disulfide
bond-reduced a-lactalbumin (a-LA), and revealed a nearly
entire reaction scheme comprising a main circular pathway
and a side pathway. In both pathways, football complexes
appear as a major intermediate. In the main pathway, which
occurs at approximately 67%, GroES alternately interacts withthe two rings of GroEL. However, in the side pathway, which
occurs at 33%, this alternate rhythm is disrupted; the second
bound GroES dissociates before the first bound GroES dissociates. The pathway branching occurs at the bullet

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

(a)

GroES

biotinylated
D490C GroEL

3

rstb.royalsocietypublishing.org

tamavidin 2-LPI
2D crystal

mica

Phil. Trans. R. Soc. B 373: 20170180(b)0s2.53 s3.45 s

4.83 s5.52 s

5.98 s6.21 s6.90 s

7.59 s8.51 s11.50 s12.19 s13.11 s16.10 s16.79 s20.47 s20 nm

Figure 1. Assay system for HS-AFM imaging of dynamic GroEL–GroES interaction and captured images. (a) Schematic of assay system used for HS-AFM imaging. Tamavidin 2-LPI was two-dimensionally crystallized directly on a bare mica surface. GroEL D490C biotinylated at Cys490 located at its equatorial domain was tethered to thetamavidin 2-LPI 2D crystal surface through the biotin–tamavidin 2-LPI linkage with a linker length of approximately 2.5 nm. Because this crystal surface is resistant tonon-specific protein binding, the tethered GroEL does not adsorb onto the surface. When the molecule is tapped briefly (approx. 100 ns) with the AFM-tip, the moleculemakes contact with the surface. During imaging, the oscillation energy of the cantilever is partially transferred to the molecule by between 2 and 3 kBT (kB, Boltzmann
constant; T, room temperature in kelvin) per tap, under the imaging conditions. This transferred energy is partitioned to many degrees of freedom of the molecule andquickly dissipates. Therefore, the tip- and surface-sample contacts do not affect the function of the molecule [40]. (b) HS-AFM images showing repeated GroES associationand dissociation at the two rings of GroEL in the presence of denatured DM-MBP. The images are clipped from successive images captured at 4.35 frames s21. The dashedlines indicate the positions of toroid ends of the GroEL molecule. The arrowheads indicate GroES bound to GroEL. Besides football and bullet complexes, GroES–unbound
GroEL also appeared at 2.53, 4.83 and 16.10 s. The bulk solution contains 1 mM GroES, 2 mM ATP and 0.2 mM denatured DM-MBP.

well fitted to single-exponential functions. All values of rate
constants estimated by single-exponential fitting are given intable 3, together with those previously obtained in the presenceof a-LA for comparison. The bullet complexes in both Type I
and Type II processes (which we refer to as Type I bullet andType II bullet, respectively) with or without DM-MBP decayedto either football complexes or GroES–unbound GroEL(figure 2), resulting in a pathway branching (i.e. B ! F or
B ! U). In addition, direct decay into bullet complexes withaltered polarity also occurred in the Type I process, irrespectiveof the substrate condition (i.e. B ! B). However, we thinkthat this event is just apparent due to occasional miscapturingof GroES–unbound GroEL, because this GroES–unbound
GroEL decays very fast into bullet complexes with opposite
polarity: 3.29 + 0.12 s21 (DM-MBP) and 8.16 + 0.17 s21(absence of SP). Because the rate constants of B ! F and B !

U transitions are approximately three-times different in theType I process and approximately two-times different in theType II process in the presence of DM-MBP (table 3), bullet
complexes are further classified into two sub-types. We referto as ‘B–F type’ and ‘B–U type’ the bullet complexes that
undergo B ! F and B ! U transitions, respectively. It would
be possible that the intermediates formed just prior to thebullet complexes might be responsible for the pathway branching and hence the sub-types. However, this is not the case,because the decay rates of these sub-types are nearly independent of the previous intermediate states, at least in the presenceof DM-MBP (electronic supplementary material, figure S3);
the bullet complexes appearing in the pathways F ! B ! F
and U ! B ! F decay at 0.96 + 0.03 and 1.00 + 0.06 s21,
respectively, while the bullet complexes appearing in thepathways F ! B ! U and U ! B ! U decay at 0.31 + 0.04

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

Table 1. Frequency (%) of appearance of intermediate species under different substrate conditions. ‘n’ indicates the total number of captured frames.
footballbulletunboundnno substrate

53.4

45.3

1.3

18 528foldable substrate

DM-MBPrhodanese

29.5
30.4

62.2
64.9

8.3
4.7

18 7595350

unfoldable substrate

a-LAa

66.8

32.8

0.4

12 714

Data from a previous study [41].

Type IaType II

BUB

BFB

BB

BUB

BFB

Type I

Type IInno substrate

6.9

52.5

13.6

1.7

25.3

73.0

27.0

1397

DM-MBPrhodanese

23.7
25.1

38.0
35.0

13.1
16.8

8.6
5.7

16.6
17.4

74.8
76.9

25.2
23.1

1287
334

a-LAa

2.4

55.7

10.7

0.4

30.8

68.8

31.2

1012

Data from a previous study [41].
(a)

(b)

footballType IabcType IIbulletunboundde

Figure 2. Three intermediate species (a) and five sequential patterns ofappearance and disappearance of these intermediate species (b) observedin the presence of foldable SP and in the absence of SP.

and 0.24 + 0.03 s21, respectively. Note that the small frequencyof observing the pathway U ! B ! U hampered precise estimation of the rate constant. In the absence of SP, the bullet
complexes appearing in the pathways F ! B ! F and U !
B! F decay at 1.26 + 0.03 and 1.03 + 0.06 s21, respectively(electronic supplementary material, figure S4). Considering
the low appearance frequency of the pathway U ! B ! F,these values are nearly identical. For the case of Type II process, we could not perform this analysis as the appearance
frequency of B ! U is small. Nevertheless, in all cases, the probabilities in which bullet complexes decay to F or U are nearly
independent of the intermediates formed just before the bullet
complexes (electronic supplementary material, table S1).
Thus, we conclude that the pathway branching occurring
after bullet complexes originates from distinct B–F and B–U

types of bullet complexes and that these sub-types are in
rapid equilibrium.
When the histograms of lifetime for bullet complexes
formed after F or U were combined, we obtained the following rate constants in the presence of DM-MBP: k BU-I ¼ 0.31 +0.02 s21, k BF-I ¼ 0.91 + 0.03 s21, k BU-II ¼ 0.53 + 0.02 s21 andk BF-II ¼ 0.97 + 0.06 s21. These values as well as thoseobtained in the absence of SP (table 3) are significantly smaller than those in the presence of a-LA (k BF-I ¼ 2.75 + 0.06 s21
and k BF-II ¼ 2.02 + 0.06 s21). Besides this prominent feature,
the appearance frequency of B –U type bullet in the presenceof foldable SP is very high (Type I B– U : Type I B –F  2 : 3),compared with the case in the absence of SP (Type I B –U :Type I B –F  1 : 7).
All histograms of lifetime for football complexes in theType I process (which we refer to as Type I football) were
well fitted to single-exponential functions, under all substrateconditions (figure 3d; electronic supplementary material,
figures S1 and S2). The estimated rate constants (k FB-I) for the
decay of F in the process B ! F ! B, which are only moderately affected by the substrate condition (approx. 0.56 s21), aregiven in table 3. On the other hand, all histograms of lifetimefor football complexes in the Type II process (which we referto as Type II football) had a peak and were well fitted to asequential two-step reaction model (figure 3h; electronic
supplementary material, figures S1 and S2). The two rate constants for the decay of F in the process B ! F ! B (k FB-II
and1
k FB-II) estimated by the fitting are given in table 3.
2
All histograms of lifetime for GroES-unbound GroEL in
the Type I process (for all substrate conditions) and Type IIprocess (only for DM-MBP) were well fitted to single-exponential functions (figure 3b,f; electronic supplementarymaterial, figures S1 and S2). The decay U ! B in the Type I
process occurs very fast (k UB-I is greater than 3.0 s21),
whereas the decay in the Type II process is much slower(k UB-II ¼ 0.92 s21 for DM-MBP).

Phil. Trans. R. Soc. B 373: 20170180

Table 2. Frequency (%) of sequential patterns in which intermediate species appear in the GroES association and dissociation reaction under different substrateconditions. The second line from the top (ﬁrst ﬁve headings) indicates sequential patterns (from left to right) of appearance and disappearance of intermediate
species (F, football; B, bullet; U, GroES – unbound GroEL). ‘n’ indicates the total number of events used in this analysis.

rstb.royalsocietypublishing.orga4

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

(a)

(b)

Type I bullet (B–U)(c)

Type I unbound
200

30

5

Type I bullet (B–F)
100

rstb.royalsocietypublishing.org

80

150

kBU-I = 0.31 ± 0.02 s–1

100

no. events

no. events

no. events

20

kUB-I = 3.29 ± 0.12 s–1

60
kBF-I = 0.91 ± 0.03 s–1

40

10
50
20
n = 305n = 2710

2

4

6 8 10 12 14 16
time (s)00(e)

Type I football
100

Phil. Trans. R. Soc. B 373: 20170180

0

0

(d)

n = 472

1

2
3
time (s)

4

5

0

(f)

Type II bullet (B–U)

1

2

3 4 5
time (s)678Type II unbound

30

30

20

20

kFB-I = 0.67 ± 0.03 s–1

40

no. events

60

no. events

no. events

80

kBU-II = 0.53 ± 0.02 s–1
10

kUB-II = 0.92 ± 0.05 s–1
1020
n = 490
0

0
0

(g)

n = 109n = 106

1

2

3 4 5
time (s)670
0 1 2 3 4 5 6 7 8 9 10
time (s)8(h)

Type II bullet (B–F)
50

0

1

2

3
4
time (s)56Type II football
30

40
250

10

200
150
100
50
0
0

1

10
n = 214n = 2090
3 4 5time (s)

6

7

8

0

GroES residence (B≠ Æ U Æ BØ Æ F Æ B≠)

6

7

8

2

4
6
time (s)810= 1.44 ± 0.04 s–1

k1
k2 = 1.25 ± 0.03 s–1
k3 = 0.91 ± 0.01 s–1

GroES residence (B≠ Æ F Æ BØ Æ F Æ B≠)

(k)

10

100
80
60
40
20
0
0

5

3 4 5
time (s)

5

30
80
60
40
20

20

0
0

5

2

4 6 8
time (s)10 12= 1.65 ± 0.30 s–1

k1
k2 = 1.20 ± 0.16 s–1
k3 = 0.34 ± 0.02 s–1

no. events

no. events

10

2cumulated number ofdissociated GroES

15

1

GroES residence (B≠ Æ F Æ BØ Æ U Æ B≠)( j)

no. events

2

cumulated number ofdissociated GroES

(i)

1

4

k1FB-II = 1.53 ± 0.29 s–1
k2FB-II = 0.83 ± 0.10 s–1

0
0

2
3
time (s)cumulated number ofdissociated GroES

kBF-II = 0.97 ± 0.06 s–1

20

cumulated number ofdecayed type II football30

no. events

no. events

20

200
150
100
50
0
0

0 1 2 3 4 5 6 7 8 9 10
time (s)810k1k2 = 1.06 ± 0.07 s–1
k3 = 0.91 ± 0.05 s–1
k4 = 0.67 ± 0.03 s–1

10

n = 810

4 6time (s)= 1.83 ± 0.21 s–1

n = 180n = 88
0

2

0
0

2

4

6 8
time (s)

10 12

0

2

4

6 8
time (s)10 12Figure 3. Histograms of lifetime of intermediate species and residence time of bound GroES observed in the presence of DM-MBP. The curves overlaid on the histograms
are those calculated using rate constants obtained by fitting. Each inset illustration in (a–k) shows a corresponding sequential pattern in which the intermediate concerned(surrounded with the red rectangle) or the bound GroES to be analysed (shown in red) appears. Value of ‘n’ attached to each histogram indicates the total number of eventsused for the lifetime and residence time analyses. The graphs inset in (h–k) show the cumulated number of corresponding events together with curves calculated using
rate constants obtained by fitting of their histograms to corresponding models (h, sequential two-step reaction; i and j, sequential three-step reaction; k, sequential fourstep reaction). For the data obtained in the absence of SP and in the presence of rhodanese, see electronic supplementary material, figures S1 and S2, respectively.

0.59 + 0.03
1.14 + 0.08
2.02 + 0.06—
0.49 + 0.04
2.75 + 0.06——

0.74 + 0.07
1.97 + 0.43
1.58 + 0.08—
0.51 + 0.03
0.97 + 0.04
4.50 + 0.10—

0.61 + 0.02
0.83 + 0.10
1.50 + 0.12
1.53 + 0.29
1.31 + 0.03
0.97 + 0.06—
0.92 + 0.05
0.56 + 0.01
0.67 + 0.03
1.21 + 0.06
0.91 + 0.03
8.16 + 0.17
3.29 + 0.12—
0.53 + 0.02

k FB-II(s21)
2
k BF-II (s21)k FB-II
(s21)
1
BFB

k FB-I (s21)k BF-I (s21)k UB-I (s21)k BU-II (s21)k UB-II (s21)

BFB
BUB

Type II

6

Here, we first describe the Type I process in the absence of SP.
When GroES binds to the trans-ring of bullet complexes, this
newly bound GroES undergoes the pathway of either (i) F !
B ! F ! B or (ii) F ! B ! U. When GroES binds to
GroES–unbound GroEL, this newly bound GroES undergoes
the pathway of either (iii) B ! F ! B or (iv) B ! U. Pathway(i) is the main pathway. Except for case (iv) where the dissociation of the bound GroEL follows a first-order reaction,
the bound GroES undergoes multiple intermediates before its
final dissociation. Here, we only analysed the histogram forthe residence time of bound GroES undergoing pathway (i)(electronic supplementary material, figure S1g) as theprobabilities of occurrence of the pathways (ii) and (iii) were
low. The histogram for the residence time of bound GroES
undergoing pathway (i) was well fitted to a sequential fourstep reaction, as was previously observed in the Type I
process in the presence of a-LA [41]. This fitting provided
k1 ¼ 1.64 s2 1, k2 ¼ 0.85 s2 1, k3 ¼ 1.20 s2 1 and k4 ¼ 0.57 s2 1.
The value of k3 is close to that of k BF-I (¼1.21 s2 1), while thevalue of k4 is close to that of k FB-I (¼0.56 s2 1). Moreover, thesum of 1/k1 and 1/k2 (1.79 s) is identical to the lifetime ofType I football (1/k FB-I ¼ 1.79 s). Therefore, we performed
the data fitting again under the restrictions of k3 ¼ k BF-I andk4 ¼ k FB-I, which provided k1 ¼ 1.84 + 0.21 s2 1 and k2 ¼
0.81 + 0.06 s2 1. The fact that 1/k1 þ 1/k2  1/k FB-I holds,
rather than 1/k1 þ 1/k2  1/k BF-I, indicates that an additional,
distinct Type I football (hereafter, we express this Type I football as F*) is formed en route to the decay of footballcomplexes to bullet complexes.
Next, we analysed the residence time distribution of bound
GroES undergoing the main pathway (i) F ! B ! F ! Bin the presence of DM-MBP (figure 3k). It was well fitted
to a sequential four-step reaction with rate constants of
k1 ¼ 1.83 + 0.21 s2 1,
k2 ¼ 1.06 + 0.07 s2 1,
k3 (¼k BF-I) ¼
21
FB-I
21
0.91 + 0.05 s
and k4 (¼ k ) ¼ 0.67 + 0.03 s . The sumof 1/k1 and 1/k2 again coincided with 1/k FB-I. For thebound GroES undergoing pathway (ii) F ! B ! U, we
obtained a moderately good fitting result, when a sequential three-step reaction was assumed: k1 ¼ 1.65 + 0.30 s2 1,
k2 ¼ 1.20 + 0.16 s2 1 and k3 ¼ 0.34 + 0.02 s2 1 ( k BU-I ¼
0.31 s21) (figure 3j ). For bound GroES undergoing pathway(iii) B ! F ! B, an excellent fitting result was obtained,
which provided k1 ¼ 1.44 + 0.04 s2 1, k2 ¼ 1.25 + 0.03 s2 1
and k3 (¼k BF-I) ¼ 0.91 + 0.01 s2 1 (figure 3i).

—a-LA

Data from a previous study [41].
a0.28 + 0.03
rhodanesea0.40 + 0.04
0.31 + 0.02
no substrate
DM-MBPk BU-I (s21)BUB

Type I

5. Discussion
In this study, we could confirm that football complexes are
not briefly subsisting but long-lived intermediates withlifetime of 1.522.0 s (Type I) or 1.922.6 s (Type II). Therefore,
football complexes must be actively involved in the chaperonin function of GroEL. However, the frequency of appearanceof football complexes moderately depends on the substratecondition, in the order of unfoldable SP (67%) . absenceof SP (53%) . foldable SP (30%). This is partly due to theshorter lifetime of football complexes in the presence offoldable SP, but mainly due to the longer lifetime (andhence more frequent appearance) of bullet complexes in thepresence of foldable SP.

Phil. Trans. R. Soc. B 373: 20170180

4. Kinetics undergone by bound GroES

rstb.royalsocietypublishing.org

Table 3. Rate constants of various reaction steps in Type I and Type II processes. The second line from the top indicates sequential patterns (from left to right) of appearance and disappearance of intermediate species (F, football;B, bullet; U, GroES – unbound GroEL).

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018(a)

DM-MBP

7

ADP–PikBF-I = 0.91 s–1

kFB-I = 0.67 s–1

rstb.royalsocietypublishing.org

ATPkBF-II = 0.97 s–1
ATP

k1 = 1.83 s–1

ATP ADPADP

ADP

kBU-I = 0.31 s–1

kBU-II = 0.53 s–1
k1FB-II = 1.53 s–1

k2 = 1.06 s–1

kUB-I = 3.29 s–1

ADP

ADP

Type I

ATP

B–U

B–F

kUB-II = 0.92 s–1
B–U

B–F

ADP

B–F

Type II

Type II
B–F
B–U

k2FB-II = 0.83 s–1
kUB-II = 0.92 s–1

B–U
Type I

ATP
ADP

ADP

kBU-II = 0.53 s–1

BU-I = 0.31 s–1
kUB-I = 3.29 s–1 ADP k

k2= 1.06 s–1

k1

kFB-II= 1.53 s–1
1

ADP
ATP ADP= 1.83 s–1
ATP

kBF-II = 0.97 s–1ATP

kFB-I = 0.67 s–1

kBF-I = 0.91 s–1
ADP–Pi(b)no substrate

ADP–PikBF-I = 1.21 s–1

kFB-I = 0.56 s–1

ATP

kBF-II = 1.31 s–1
ATP

k1 = 1.84 s–1

ATP ADPADP

ADP

kBU-I = 0.40 s–1

rare

k1FB-II = 1.50 s-1

k2 = 0.81 s–1

kUB-I = 8.16 s–1

ADP

ADP

Type IATP

kFB-II= 0.61 s–1
2

B–U

B–F

ADP

B–U

B–F

Type II

B–F

B–U
Type I

ADP

Type II
B–F
B–U

kFB-II= 0.61 s–1
2

ATP
ADP

ADP

kUB-I = 8.16 s–1
k2 = 0.81 s–1

ADP

k1FB-II = 1.50 s–1

rare

kBU-I = 0.40 s–1

ADP
ATP ADPk1 = 1.84 s–1

ATP

kBF-II = 1.31 s–1
ATP

kFB-I = 0.56 s–1

(c)

a-LA

ADP–PikBF-I = 1.21 s–1

ADP–PikBF-I = 2.75 s–1

kFB-I = 0.49 s–1

ATP

kBF-II = 2.02 s–1ATP

k1 = 1.14 s–1

ATP ADP
ADP

rare

rare

k1FB-II = 1.14 s–1

k2 = 0.87 s–1

ADP

ADP

ADP

Type IATP

kFB-II= 0.59 s–1
2

B–U
B–F
Type II

ADP

B–U

B–F

B–F

B–U
Type I

ADP

Type II
B–F
B–U

k2FB-II = 0.59 s–1

ATP
ADP

ADP

rare

ADP

ADP

k2

= 0.87 s–1

k1

k1FB-II = 1.14 s–1

rare
ATP ADP= 1.14 s–1
ATP

kBF-II = 2.02 s–1ATP

kFB-I = 0.49 s–1

kBF-I = 2.75 s–1
ADP–PiFigure 4. Kinetic reaction schemes of GroEL–GroES interaction revealed by HS-AFM imaging under different substrate conditions. The football complexes (F*) shown inpale colours are apparently the same as but kinetically different from the football complexes formed immediately before. The thickness of arrows (shown with solid black,
red, pink and green lines) along reaction pathways indicates the relative frequency of occurrence. The dashed red arrows indicate reaction processes occurring in the ring
FB-IIbound to GroES shown in red, while the dashed blue arrow indicates reaction processes occurring in the ring bound to GroES shown in blue. The order of k FB-II
1 and k 2 andthe order of k1 and k2 are tentative. In the side pathway, the coexistence of ATP and ADP in one ring is shown but hypothetical. (a) Scheme of kinetic reaction in thepresence of DM-MBP. (b) Scheme of kinetic reaction in the absence of SP. (c) Scheme of kinetic reaction in the presence of a-LA.

Phil. Trans. R. Soc. B 373: 20170180

k2FB-II = 0.83 s–1

ADP

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 20188

Phil. Trans. R. Soc. B 373: 20170180

here (2 mM) is nearly saturating, and the concentration of
GroES (1 mM) is also nearly saturating, considering thesecond-order rate constant for GroES binding (1– 3 
107 M21 s21) [44]. Therefore, this transition rate k BF-I is identical to the rate of ADP release from the trans-ring, indicatingthat ADP remains bound for a while in the trans-ring, consistent with biochemical observations [45,46]. However, toalleviate the discrepancies regarding the presence of footballcomplexes, an idea has been proposed that this ADP releaseretardation would occur only in the absence of SP and thisnegative cooperativity could be weakened in the presenceof SP. Therefore, bullet complexes are accumulated in theabsence of SP, whereas in the presence of SP the accelerated
ADP dissociation and hence the accelerated ATP/GroES binding [27,45] leads to the formation of football complexes [26,28].
However, this idea is inconsistent with our observations.
The value of k BF-I in the absence of SP (1.21 s21) is insteadsomewhat larger than that in the presence of foldable SP(0.91 –0.97 s21). Moreover, football complexes are formed in
substantial amount (approx. 50%), even in the absence of SP.
In our previous study [41], we proposed that the asymmetricbullet structure would cause a strain in the molecule andthis strain would retard ADP release from the cis-ring, likethe retardation of ADP release from the leading head of
myosin V walking on actin filaments [39,47]. What is occurringin the cis-ring during this B ! F transition? One possibility is
ATP hydrolysis to ADP –Pi. The hydrolysis rate in the presenceof DM-MBP calculated based on this assumption, 1/(1/k FB-I þ
1/k BF-I) ¼ 0.39 s21, is close to the biochemically estimatedvalues, 0.3120.36 s21. If this reaction does occur in the transition step, the two rings in the bullet complexes mutuallycommunicate to suppress ATP hydrolysis in the cis-ring and
ADP release from the trans-ring. Compared with the case of
DM-MBP, the strength of this negative cooperativity is 30%
weaker in the absence of SP and 200% weaker in the presenceof a-LA, judging from the values of k BF-I. This negative cooperativity ensures the release of SP from the trans-ring before
this ring is capped with GroES. Another interpretation for
k3 ¼ k BF-I would be that the substrate-dependent ATP hydrolysis into ADP –Pi triggers ADP release from the opposite ring.
The additional coincidence of 1/k1 þ 1/k2 ¼ k FB-I also indicates an allosteric communication between the two rings.
However, the ambiguity about physical or chemical eventsoccurring in the transition steps corresponding to k1 and k2
prevents us from gaining insights into the nature of thiscommunication. Nevertheless, we think that ATP-bindinginduced conformational change occurring in the second
cis-ring in football complexes triggers Pi release and hence
GroES release from the opposite ring.
Finally, we discuss the Type II process. Type II bullet distinct from Type I bullet is responsible for the Type II process.
The allosteric communication between the two rings in TypeI bullet (corresponding to the coincidence of k3 ¼ k BF-I) must
be disrupted in the Type II process. In our previous HS-AFM
study in the presence of a-LA, we proposed that the Type IIprocess would occur due to incomplete nucleotide exchange(unlike the replacement of seven ADPs by seven ATPs) at thetrans-ring of bullet complexes [41]. The disruption of the allosteric communication possibly causes partial ADP releasefrom the trans-ring without ATP hydrolysis in the cis-ring.
The reduced number of bound ATP in the new cis-ring must
only induce a small conformational change in this ring,which would not be large enough to affect the opposite ring.

rstb.royalsocietypublishing.org

The entire reaction pathway of the GroE system revealed byits direct observation with HS-AFM is much more complicatedthan previously realized, and also highly stochastic as with thepreviously suggested partial stochasticity of the ATPase reaction in the GroE system [28]. This complexity first results fromthe two distinct pathways, Type I and Type II. In the latter,
the alternate rhythm of binding and release of GroES at thetwo rings is disrupted, which often occurs (25–33%), independently of the substrate condition. Second, GroES–unbound
GroEL sometimes appears after bullet complexes, especiallyin the presence of foldable SP (5–8%). The resulting pathway
branching (either B ! F or B ! U) occurs independently of
the intermediates (F or U) formed just before the bullet complexes. Therefore, there must be two sub-types of bullet
complexes (B–F type and B–U type) in rapid equilibrium, inboth Type I and Type II processes.
From the observed patterns of sequential GroES binding
and release and the analysis of lifetime of all intermediates
in each pathway and residence time of GroES, we constructeda model for the reaction scheme under each substrate condition, as shown in figure 4. The scheme in the presence ofa-LA is the one modified from our previous study [41].
These schemes are partially tentative because several
issues in this extremely complicated reaction are still open
to clarification, as described below.
Although it is clear that F* occurs en route to the decay of
F ! B, this decay occurs twice in the main pathway of F ! B !
F ! B. Therefore, there are two possibilities: F ! F* ! B !
F ! B or F ! B ! F ! F* ! B. If the former is the case,F* must originate in the newly formed (second) cis-ring of
the first football complexes, rather than in the early formed(first) cis-ring of the first football complexes. One possibility
is that F* is formed by an ATP-binding-induced conformational change in the second cis-ring (apical domain movement
or a conformational change that can drive SP encapsulation).
If F* is formed in the sequence F ! B ! F ! F* ! B, one
possibility is that ATP hydrolysis to ADP–Pi in the first cisring of the second football complexes induces the formationof F*. In this case, the rate of ATP hydrolysis into ADP–Pi in
the presence of DM-MBP is 1/(1/k FB-I þ 1/k BF-I þ 1/k1) ¼
0.32 s21 or 1/(1/kFB-I þ 1/kBF-I þ 1/k2) ¼ 0.28 s21. These values
are close to the values estimated from biochemical studies in
the presence of foldable SP, 0.3120.36 s21 [35,43]. Note thatthe biochemically determined rate of ATP ! ADP2Pi is anaverage over those in different pathways. Likewise, in themain pathway in the absence of SP, the rate of ATP hydrolysis
into ADP2Pi can be estimated to be either 0.32 or 0.26 s21,
again close to the reported values. In spite of these good agreements, we think that this is not the case because this model
suggests a sequential two-step reaction for the final dissociationof bound GroES, contrary to the observed single-exponential
distribution of Type I football. Therefore, it is very likely that
F* originates from an ATP-binding-induced conformationalchange in the second cis-ring of football complexes.
Next, we discuss allosteric communications between thetwo rings of GroEL in the main circular pathway. The residence time analysis of bound GroES showed the coincidenceof k3 ¼ k BF-I, which indicates that the bound GroES on the
cis-ring senses a certain change occurring in the trans-ringand vice versa. In the presence of SP, this B ! F transition isaccompanied by a series of events in the trans-ring: the ejectionof SP, release of ADP, binding of new ATP and then GroES
binding to the trans-ring. The concentration of ATP used

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

Data accessibility. This article has no additional data.
Authors’ contributions. T.A. designed this study, developed the highspeed AFM instrument and mathematical methods to analyse the histograms of residence time of bound GroES and wrote the manuscript.
D.N. prepared all samples used in this study, performed all high-speed AFM imaging experiments, and prepared all figures andtables. The final manuscript was prepared through discussions
between T.A. and D.N.
Competing interests. We have no competing interests.

Funding. This work was supported by grants to T.A.: KAKENHI,
Grants-in-Aid for Scientific Research (S) (no. 24227005 and no.
17H06121); KAKENHI, Grants-in-Aid for Research on Innovative
Area (research in a proposed research area; no. 26119003) andCREST programme of the Japan Science and Technology Agency(no. JPMJCR13M1). This work was also supported in part by
KAKENHI, Young Scientists (B) (no. 17K15433 to D.N.).

1.

Hartl FU, Bracher A, Hayer-Hartl M. 2011
Molecular chaperones in protein folding andproteostasis. Nature 475, 324–332. (doi:10.1038/
nature10317)
2. Hartl FU, Hayer-Hartl M. 2002 Molecular chaperonesin the cytosol: from nascent chain to folded protein.
Science 295, 1852 –1858. (doi:10.1126/science.
1068408)
3. Braig K, Otwinowski Z, Hegde R, Boisvert DC,
Joachimiak A, Horwich AL, Sigler PB. 1994 The
crystal structure of the bacterial chaperonin GroEL
at 2.8 Å. Nature 371, 578–586. (doi:10.1038/
371578a0)
4. Hunt JF, Weaver AJ, Landry S, Gierasch L,
Deisenhofer J. 1996 The crystal structure of the
GroES co-chaperonin at 2.8 Å resolution. Nature
379, 37 –45. (doi:10.1038/379037a0)
5. A Horwich AL, Fenton WA. 2009 Chaperoninmediated protein folding: using a central cavity tokinetically assist polypeptide chain folding. Q. Rev.
Biophys. 42, 83– 116. (doi:10.1017/
S0033583509004764)
6. Chaudhuri TK, Verma VK, Maheshwari A. 2009
GroEL assisted folding of large polypeptide
substrates in Escherichia coli: present scenario
and assignments for the future. Prog. Biophys.
Mol. Biol. 99, 42 –50. (doi:10.1016/j.pbiomolbio.
2008.10.007)
7. Jewett AI, Shea JE. 2010 Reconciling theories ofchaperonin accelerated folding with experimental
evidence. Cell. Mol. Life Sci. 67, 255– 276. (doi:10.
1007/s00018-009-0164-6)
8. Clare DK, Bakkes PJ, van Heerikhuizen H, van derVies SM, Saibil HR. 2009 Chaperonin complex with a
newly folded protein encapsulated in the folding
chamber. Nature 457, 107–110. (doi:10.1038/
nature07479)
9. Weissman JS, Hohl CM, Kovalenko O, Kashi Y, Chen
S, Braig K, Saibil HR, Fenton WA, Norwich AL. 1995
Mechanism of GroEL action: productive release ofpolypeptide from a sequestered position under
GroES. Cell 83, 577–587. (doi:10.1016/0092-8674(95)90098-5)
10. Mayhew M, da Silva AC, Martin J, ErdjumentBromage H, Tempst P, Hartl FU. 1996 Protein
folding in the central cavity of the GroEL-GroES
chaperonin complex. Nature 379, 420– 426. (doi:10.
1038/379420a0)

11. Yifrach O, Horovitz A. 1995 Nested cooperativity in
the ATPase activity of the oligomeric chaperonin
GroEL. Biochemistry 34, 5303–5308. (doi:10.1021/
bi00016a001)
12. Burston SG, Ranson NA, Clarke AR. 1995 The origins
and consequences of asymmetry in the chaperonin
reaction cycle. J. Mol. Biol. 249, 138–152. (doi:10.
1006/jmbi.1995.0285)
13. Horovitz A, Willison KR. 2005 Allosteric regulation ofchaperonins. Curr. Opin. Struct. Biol. 15, 646–651.
(doi:10.1016/j.sbi.2005.10.001)
14. Xu Z, Horwich AL, Sigler PB. 1997 The crystal
structure of the asymmetric GroEL-GroES-(ADP)7chaperonin complex. Nature 388, 741 –750. (doi:10.
1038/41944)
15. Rye HS, Burston SG, Fenton WA, Beechem JM, Xu Z,
Sigler PB, Horwich AL. 1997 Distinct actions of cis andtrans ATP within the double ring of the chaperonin
GroEL. Nature 388, 792–798. (doi:10.1038/42047)
16. Rye HS, Roseman AM, Chen S, Furtak K, Fenton WA,
Saibil HR, Horwich AL. 1999 GroEL-GroES cycling:
ATP and nonnative polypeptide direct alternationof folding-active rings. Cell 97, 325 –338. (doi:10.
1016/S0092-8674(00)80742-4)
17. Schmidt M, Rutkat K, Rachel R, Pfeifer G, Jaenicke
R, Viitanen P, Lorimer G, Buchner J. 1994
Symmetric complexes of GroE chaperonins as part of
the functional cycle. Science 265, 656 –659. (doi:10.
1126/science.7913554)
18. Azem A, Kessel M, Goloubinoff P. 1994
Characterization of a functional GroEL14(GroES7)2
chaperonin hetero-oligomer. Science 265, 653–656.
(doi:10.1126/science.7913553)
19. Llorca O, Marco S, Carrascosa JL, Valpuesta JM. 1994
The formation of symmetrical GroEL-GroES
complexes in the presence of ATP. FEBS Lett. 345,
181 –186. (doi:10.1016/0014-5793(94)00432-3)
20. Beißinger M, Rutkat K, Buchner J. 1999 Catalysis,
commitment and encapsulation during GroEmediated folding. J. Mol. Biol. 289, 1075– 1092.
(doi:10.1006/jmbi.1999.2780)
21. Azem A, Diamant S, Kessel M, Weiss C, Goloubinoff,
P. 1995 The protein-folding activity of chaperonins
correlates with the symmetric GroEL14(GroES7)2
heterooligomer. Proc. Natl Acad. Sci. USA 92,
12 021– 12 025. (doi:10.1073/pnas.92.26.12021)
22. Sparrer H, Rutkat K, Buchner J. 1977 Catalysis ofprotein folding by symmetric chaperone complexes.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Proc. Natl Acad. Sci. USA 94, 1096–1100. (doi:10.
1073/pnas.94.4.1096)
Koike-Takeshita A, Arakawa T, Taguchi H,
Shimamura T. 2014 Crystal structure of a symmetricfootball-shaped GroEL:GroES2-ATP14 complex
determined at 3.8 Å reveals rearrangement between
two GroEL rings. J. Mol. Biol. 426, 3634–3641.
(doi:10.1016/j.jmb.2014.08.017)
Taguchi H. 2015 Reaction cycle of chaperonin
GroEL via symmetric ‘football’ intermediate. J. Mol.
Biol. 427, 2912– 2918. (doi:10.1016/j.jmb.2015.
04.007)
Koike-Takeshita A, Yoshida M, Taguchi H. 2008
Revisiting the GroEL-GroES reaction cycle via thesymmetric intermediate implied by novel aspectsof the GroEL(D398A) mutant. J. Biol. Chem. 283,
23 774 –23 781. (doi:10.1074/jbc.M802542200)
Sameshima T, Iizuka R, Ueno T, Funatsu T. 2010
Denatured proteins facilitate the formation of thefootball-shaped GroEL-(GroES)2 complex. Biochem. J.
427, 247–254. (doi:10.1042/BJ20091845)
Ye X, Lorimer GH. 2013 Substrate protein switches
GroE chaperonins from asymmetric to symmetric
cycling by catalyzing nucleotide exchange. Proc.
Natl Acad. Sci. USA 110, E4289–E4297. (doi:10.
1073/pnas.1317702110)
Yang D, Ye X, Lorimer GH. 2013 Symmetric
GroEL:GroES2 complexes are the protein-folding
functional form of the chaperonin nanomachine.
Proc. Natl Acad. Sci. USA 110, E4298– E4305.
(doi:10.1073/pnas.1318862110)
Fei X, Ye X, LaRonde NA, Lorimer GH. 2014
Formation and structures of GroEL:GroES2
chaperonin footballs, the protein-folding functional
form. Proc. Natl Acad. Sci. USA 111, 12 775–
12 780. (doi:10.1073/pnas.1412922111)
Rye HS. 2001 Application of fluorescence resonanceenergy transfer to the GroEL-GroES chaperonin
reaction. Methods 24, 278 –288. (doi:10.1006/
meth.2001.1188)
Hosono K, Ueno T, Taguchu H, Motojima F, Zako T,
Yoshida M, Funatsu T. 2008 Kinetic analysis ofconformational changes of GroEL based on the
fluorescence of tyrosine 506. Protein J. 27,
461–468. (doi:10.1007/s10930-008-9157-9)
Takei Y, Iizuka R, Ueno T, Funatsu T. 2012 Singlemolecule observation of protein folding insymmetric GroEL-(GroES)2 complexes. J. Biol. Chem.

Phil. Trans. R. Soc. B 373: 20170180

References9

rstb.royalsocietypublishing.org

This is consistent with the fact that the early bound GroES
never dissociates in the Type II process. Nevertheless, several
issues concerning the Type II process are still open to clarification. Since this process occurs to a non-negligible extent(25 –33%), further studies are awaited on this process.

Downloaded from http://rstb.royalsocietypublishing.org/ on May 7, 2018

34.

36.

37.

39.

40.

41.

42.

43. Suzuki M et al. 2008 Effect of the C-terminal
truncation on the functional cycle of chaperonin
GroEL: implication that the C-terminal region
facilitates the transition from the folding-arrested
to the folding-competent state. J. Biol. Chem. 283,
23 931 –23 939. (doi:10.1074/jbc.M804090200)
44. Motojima F, Chaudhry C, Fenton WA, Farr GW,
Horwich AL. 2004 Substrate polypeptide presents
a load on the apical domains of the chaperonin
GroEL. Proc. Natl Acad. Sci. USA 101, 15 005 –15 012. (doi:10.1073/pnas.0406132101)
45. Grason JP, Gresham JS, Widjaja L, Wehri SC,
Lorimer GH. 2008 Setting the chaperonintimer: the effects of Kþ and substrate
protein on ATP hydrolysis. Proc. Natl Acad. Sci.
USA 105, 17 334–17 338. (doi:10.1073/pnas.
0807429105)
46. Madan D, Lin Z, Rye HS. 2008 Triggering protein
folding within the GroEL-GroES complex, J. Biol.
Chem. 283, 32 003–32 013. (doi:10.1074/jbc.
M802898200)
47. Purcell TJ, Sweeney HL, Spudich JA. 2005 A forcedependent state controls the coordination ofprocessive myosin V. Proc. Natl Acad. Sci. USA 102,
13 873 –13 878. (doi:10.1073/pnas.0506441102)

10

Phil. Trans. R. Soc. B 373: 20170180

35.

38.

Acad. Sci. USA 98, 12 468–12 472. (doi:10.1073/
pnas.211400898)
Ando T, Uchihashi T, Fukuma T. 2008 Highspeed atomic force microscopy for nanovisualization of dynamic biomolecular processes.
Prog. Surf. Sci. 83, 337–437. (doi:10.1016/j.
progsurf.2008.09.001)
Kodera N, Yamamoto D, Ishikawa R, Ando T. 2010
Video imaging of walking myosin V by high-speed
atomic force microscopy. Nature 468, 72 –76.
(doi:10.1038/nature09450)
Ando T, Uchihashi T, Scheuring S. 2014 Filming
biomolecular processes by high-speed atomic force
microscopy. Chem. Rev. 114, 3120 –3188. (doi:10.
1021/cr4003837)
Yamamoto D, Ando T. 2016 Chaperonin GroEL-GroES
functions as both alternating and non-alternating
engines. J. Mol. Biol. 428, 3090 –3101. (doi:10.
1016/j.jmb.2016.06.017)
Takakura Y, Oka N, Kajiwara H, Tsunashima M.
2012 Engineering of novel tamavidin 2 muteins
with lowered isoelectric points and lowered
non-specific binding properties. J. Biosci. Bioeng.
114, 485 –489. (doi:10.1016/j.jbiosc.2012.
06.009)rstb.royalsocietypublishing.org

33.

287, 41 118–41 125. (doi:10.1074/jbc.M112.
398628)Sameshima T et al. 2010 Single-molecule study on
the decay process of the football-shaped GroELGroES complex using zero-mode waveguides. J. Biol.
Chem. 285, 23 159 –23 164. (doi:10.1074/jbc.M110.
122101)
Taguchi H, Ueno T, Tadakuma H, Yoshida M,
Funatsu T. 2001 Single-molecule observation ofprotein –protein interactions in the chaperoninsystem. Nat. Biotechnol. 19, 861– 865. (doi:10.
1038/nbt0901-861)
Ueno T, Taguchi H, Tadakuma H, Yoshida M,
Funatsu T. 2004 GroEL mediates protein folding
with a two successive timer mechanism. Mol.
Cell 14, 423–434. (doi:10.1016/S10972765(04)00261-8)
Haldar S, Gupta AJ, Yan X, Miličić G, Hartl FU,
Hayer-Hartl M. 2015 Chaperonin-assisted protein
folding: relative population of asymmetric andsymmetric GroEL:GroES complexes. J. Mol. Biol. 427,
2244–2255. (doi:10.1016/j.jmb.2015.04.009)
Ando T, Kodera N, Takai E, Maruyama D, Saito K,
Toda A. 2001 A high-speed atomic force microscopefor studying biological macromolecules. Proc. Natl