article

published online: 17 March 2013 | doi: 10.1038/nchembio.1213

A single-molecule dissection of ligand binding to a

protein with intrinsic dynamics
Eunkyung Kim1,8, Sanghwa Lee2,3,8, Aram Jeon4,5, Jung Min Choi1, Hee-Seung Lee4,5, Sungchul Hohng2,3,6* &
Hak-Sung Kim1,7*

Protein dynamics have been suggested to have a crucial role in biomolecular recognition, but the precise molecular mechanisms
remain unclear. Herein, we performed single-molecule fluorescence resonance energy transfer measurements for wild-type
maltose-binding protein (MBP) and its variants to demonstrate the interplay of conformational dynamics and molecular recog-
nition. Kinetic analysis provided direct evidence that MBP recognizes a ligand through an ‘induced-fit’ mechanism, not through
the generally proposed selection mechanism for proteins with conformational dynamics such as MBP. Our results indicated
that the mere presence of intrinsic dynamics is insufficient for a ‘selection’ mechanism. An energetic analysis of ligand binding
implicated the critical role of conformational dynamics in facilitating a structural change that occurs upon ligand binding.

T
© 2013 Nature America, Inc. All rights reserved.

he function of proteins depends on the recognition and bind-
ing of specific ligands. Recent studies have revealed that many
proteins exist in different structural substates even without
the existence of cognate ligands1–3, which suggests that conforma-
tional dynamics has prominent roles in many biological processes,
including molecular recognition4–8. The presence of conformational
substates has implicated a linkage between protein dynamics and
molecular recognition, leading to the development of dynamics-
coupled recognition mechanisms9–11. In the induced-fit model12–15,
a ligand binds a highly populated open conformation of a protein,
followed by a structural adjustment to a ligand-bound, fully closed
state (Fig. 1a). In contrast, the conformational selection mechanism
assumes that a ligand selectively binds a weakly populated single
substate in the ensemble of conformations, and no further confor-
mational change occurs after ligand binding (Fig. 1a). In several
cases, it has been shown that apo proteins sample a partially closed
form that resembles the ligand-bound form but can be distinguished
from it by fine details. It has therefore been suggested that the con-
formational selection and induced-fit mechanisms are not mutu-
npg
in a time-resolved manner at the single-molecule level19,20. Unlike
other experimental methods, single-molecule fluorescence reso-
nance energy transfer (smFRET) measurements enable a kinetic
analysis of the conformational dynamics of proteins. A comparison
of the opening and closing kinetics in the absence and presence
of varying ligand concentrations provides crucial insight into the
molecular recognition process. As simply depicted in Figure 1b,
for the induced-fit model, the closing rate is expected to acceler-
ate with an increase in the ligand concentration, whereas no change
in the closing rate is observed with the conformational selection
mechanism. However, such a simple scheme has never been realized
because intrinsic conformational dynamics are too rare and brief to
be directly observed through smFRET.
Herein, we demonstrate the connection between the confor-
mational dynamics and molecular recognition of proteins in the
process of ligand binding through a real-time kinetic analysis using
smFRET measurements. A wild-type MBP and its variants were
used in this study. Ligand binding of MBP, which is involved in
the active transport of maltodextrins in Escherichia coli, has been

ally exclusive but are cooperative for the molecular recognition
process16–18. In the extended conformational selection model, which
suggests a more general framework, the conformational selection
mechanism works for the initial selective binding of a ligand to a
partially closed form and is followed by a subsequent fine structural
adjustment through an induced-fit mechanism.
Despite the evidence for protein dynamics and conceptual
advances, however, molecular mechanisms that drive ligand recogni-
tion and the role of conformational dynamics in the molecular recog-
nition of proteins remain controversial and elusive mainly owing to
experimental limitations. To truly understand the interplay between
protein dynamics and the molecular recognition mechanism, an
experimental method to directly correlate the binding of ligands to
the conformational dynamics of proteins in real time is necessary. In
this respect, a single-molecule analysis offers the potential to resolve
the controversy by detecting transient ­intermediates or rare events
considered a classic example of the induced-fit model because of
the structural differences between its ligand-free and ligand-bound
forms21. However, recent paramagnetic NMR experiments and
molecular dynamics simulations have revealed that a ligand-free
MBP undergoes dynamic structural transitions between a major
open form and a minor partially closed form22,23, raising an issue
in the ligand recognition process. A previous study reported that
ligands are accessible to the sugar-binding surface of the C-terminal
domain in the partially closed conformation of an apo wild-type
MBP22. It has therefore been suggested that ligands might pref-
erentially bind the partially closed form of an MBP through a
conformational selection mechanism, followed by fine confor-
mational adjustment through the induced-fit mechanism4. We
explored the molecular recognition mechanism of an MBP on the
basis of a kinetic analysis of the conformational dynamics using
smFRET measurements.

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea. 2Department of Physics and Astronomy, Seoul
1

National University, Seoul, Korea. 3National Center for Creative Research Initiatives, Seoul National University, Seoul, Korea. 4Department of Chemistry,
Korea Advanced Institute of Science and Technology, Daejeon, Korea. 5Molecular-Level Interface Research Center, Korea Advanced Institute of Science and
Technology, Daejeon, Korea. 6Biophysics and Chemical Biology, Seoul National University, Seoul, Korea. 7Graduate School of Nanoscience and Technology,
Korea Advanced Institute of Science and Technology, Daejeon, Korea. 8These authors contributed equally to this work. *e-mail: shohng@snu.ac.kr or
hskim76@kaist.ac.kr

nature CHEMICAL BIOLOGY | vol 9 | MAY 2013 | www.nature.com/naturechemicalbiology 313

 article
RESULTS
Intrinsic dynamics of MBPs
To investigate the intrinsic dynamics of MBP in the absence of a
ligand using smFRET, we first labeled wild-type MBP with a biotin
at its N terminus and with a FRET pair (Cy3 and Cy5) on the internal
residues. We selected the dye-labeling positions (Lys34 and Arg354)
such that distinct changes in FRET efficiency arise during the con-
formational transitions between the apo and holo forms (Fig. 2).
Both a CD analysis and an amylose-binding assay confirmed that
the effect of cysteine introduction and dye labeling on the biological
activity of wild-type MBP was negligible (Supplementary Results,
Supplementary Fig. 1). To observe the real-time conformational
dynamics of the proteins, we immobilized dye-labeled MBPs on
a PEG-coated quartz surface through a streptavidin-biotin inter-
action (Supplementary Fig. 2) and imaged using a total internal
reflection fluorescence microscope (Supplementary Fig. 3). As the
paramagnetic NMR experiments revealed, a ligand-free wild-type
MBP is expected to be in dynamic equilibrium between a major
open state (~95%) and a weakly populated partially closed state
(~5%)22. However, we could not detect such fast conformational
dynamics in the ns-μs range22 through smFRET analysis with a
15-ms time resolution (Fig. 3a). In an effort to increase the time
resolution, we conducted smFRET measurements using a 128 pixel
© 2013 Nature America, Inc. All rights reserved.
× 128 pixel electron-multiplying charge-coupled device camera with
a 2-ms time resolution. Nonetheless, no distinct transitions between
the two substates were detected for wild-type MBP in the absence or
presence of maltose (Supplementary Fig. 4).
It has been previously shown that mutations at the hinge region
of MBP affect its conformational equilibrium and ligand binding
affinity24,25; the mutants in this region had greatly increased ligand
binding affinities compared to the wild type, mainly because of the
changes in the off rates. In addition, the binding affinities of the
Ile329 mutants for maltose show a strong correlation with the side
chain volume of the amino acid at position 329 (ref. 24). The bind-
ing affinity of maltose for an I329W mutant, whose side chain is
larger than that of the I329F mutant but smaller than that of I329C-
thionitropyridine (I329C-TNP), lies between those measured for
I329F and I329C-TNP24. On the basis of these findings, we expected
that introduction of bulky amino acids into the hinge region would
facilitate a kinetic analysis using smFRET by slowing down the
intrinsic conformational dynamics. Specifically, we attempted to use
single (I329W) and double (A96W I329W) mutants of the MBP. We
npg
constructed the I329W and A96W I329W mutants with two cysteine
mutations, K34C and R354C, for dual dye labeling as described above
for wild-type MPB. The CD spectra of the wild type and the mutants
were almost identical (Supplementary Fig. 5), indicating negligible
differences in their secondary structures. In comparison to the wild
type, which has a maltose dissociation constant of 2,180 ± 190 nM
(mean ± s.d.), the mutants showed increased ligand binding affini-
ties, as determined through isothermal titration calorimetry (ITC):
48 ± 19 nM for the single mutant (I329W) and 4.7 ± 3.3 nM for
the double mutant (A96W I329W; Table 1). We tested whether the
intrinsic conformational dynamics of the mutants could be detected
in the absence of maltose, and we were able to clearly observe the
transitions between the two distinct conformational states (Fig. 3b,c).
The low- and high-FRET states were interpreted as open and par-
tially closed forms, respectively. We estimated the partially closed
states of the single and double mutants to be 14% and 42% of the
total populations, respectively (Fig. 3d). A kinetic analysis revealed
that the closing and opening rates of the double mutant are much
slower than those of the single mutant (Fig. 3e,f).
Effects of ligand on the kinetic rates of MBPs
We next examined the ligand recognition process of MBPs in con-
nection with intrinsic dynamics. A kinetic analysis in the absence
and presence of a ligand provides direct evidence for the ­interplay
314
Nature chemical biology doi: 10.1038/nchembio.1213
a Induced fit
Po L Po-L
+
+
Pc L Pc-L
Conformational
selection
b No ligand
Tclosing
FRET
Conformational selection Induced fit
+ Ligand + Ligand
FRET
FRET
++ Ligand ++ Ligand
FRET
FRET
Time Time
Figure 1 | Molecular recognition processes for induced-fit and conformational
selection mechanisms. (a) Thermodynamic cycles for conformational
selection and induced-fit mechanisms. Po, Pc and L indicate the protein’s
open conformation, closed conformation and a ligand, respectively. (b) The
expected time traces of the FRET efficiency in smFRET measurements for
the respective recognition mechanisms. Tclosing indicates the time for a protein
to undergo a conformational change from an open to a closed state in the
absence or presence of varying ligand concentrations. Plus symbols represent
the ligand concentration. Arrowheads indicate the binding of a ligand.
of conformational dynamics and the molecular recognition
mechanism of proteins4,26. If the ligands bind a highly populated
open form and induce a conformational transition of the protein
to a closed form, the closing rates will be faster in the presence of
a ligand than in its absence and will be accelerated by increasing
ligand concentration (Fig. 1b). In contrast, if the ligands selectively
bind a weakly populated, partially closed conformation, the clos-
ing rates in the presence of a ligand will be the same as those with
no ligand because the population of a ligand-binding conformer
is limited by the intrinsic conformational transition rate (Fig. 1b).
Therefore, to demonstrate the molecular recognition mechanism,
the intrinsic protein dynamics and the dependence of the closing
rate on the ligand concentration should be determined. Figure 4a
shows the representative intensity and FRET time traces for the
single (I329W) mutant at varying maltose concentrations. The
high-FRET state of the single mutant became more populated with
increasing maltose concentrations.
It was noticeable that the ligand-free partially closed form
and the ligand-bound closed form were indistinguishable in our
smFRET assay. To verify that the FRET changes came from the
conformational transitions between the two substates as predicted
by the crystal structures, we attempted to determine the inter-dye
distances on the basis of the FRET efficiencies. Although no crys-
tal structure of the single mutant is available, it was reported that
mutation of the single mutant most likely predominantly affects
the ligand-free state of the protein, suggesting a closure angle of
nature chemical biology | vol 9 | MAY 2013 | www.nature.com/naturechemicalbiology
 Nature chemical biology doi: 10.1038/nchembio.1213 article
K34C volume simulation. In the simulation, we used geometric constraints
K34C
based on the structural data for ligand-free and ligand-bound wild-
type MBPs and physical properties of dyes and flexible linkers (for
example, 1 nm for linker length, 0.5 nm for linker diameter and
0.7 nm for dye diameter). The inter-dye distances from the mea-
sured FRET efficiencies were comparable to those measured by the
­probability-based analysis method, supporting the conclusion that the

5.04 nm
5.92 nm
A96 A96
I329
I329
FRET changes in our study reflected the conformational transitions
between the two substates as predicted by the crystal structures.
The kinetic analysis of the closing and opening transitions for
the single mutant at varying ligand concentrations revealed that
Maltose the closing rate was faster in the presence of the ligand than in its
R354C absence and that this rate increased linearly with an increase in the
R354C
ligand concentration (Fig. 4b and Supplementary Fig. 7a). In con-
trast, the opening rates remained almost constant over the range of
Figure 2 | Experimental design for analyzing the conformational
tested ligand concentrations or in the absence of the ligand (Fig. 4c
dynamics of MBP. Structures of E. coli MBPs (Protein Data Bank codes
and Supplementary Fig. 7b). A similar behavior was observed with
1OMP (ligand-free, cyan) and 1ANF (ligand-bound, yellow)). The two
maltotriose (Fig. 4b,c and Supplementary Fig. 8) but not with
cysteine mutations (K34C, R354C) introduced for conjugating the dyes
sucrose, which does not bind MBP (Fig. 4b,c and Supplementary
are indicated. The distances between the Cα atoms of these residues in
the apo and holo proteins were estimated to be 5.92 nm and 5.04 nm,
Fig. 9). To test whether our smFRET analysis reliably monitors single
respectively. The mutation sites for the construction of single (A96W) and
ligand binding events, we determined the dissociation constant (Kd)
double (A96W I329W) mutants are also shown.
on the basis of the probability of the high FRET state (Online Methods
and Supplementary Fig. 10a–c). In the single mutant, we obtained
© 2013 Nature America, Inc. All rights reserved.

the dissociation constants of 99 nM for maltose and 56 nM for

9.5° ± 0.9° (mean ± s.d.) compared to wild-type MBP, with a subtle
change in the structure of the ligand-bound state25. It is therefore
likely that both conformers of the single mutant will not differ from
those of wild-type MBP. Our previous study estimated the Förster
distance (R0) for a Cy3-Cy5 FRET pair to be 5.4 nm (ref. 27). Using
the R0 value and FRET efficiencies in the presence of 100 nM malt-
ose for the MBP single mutant (Supplementary Fig. 6), we deter-
mined the inter-dye distances for both conformers (Supplementary
Table 1). We also calculated the inter-dye distances through a
probability-based analysis method using the Model Satellite Prior
Algorithm28 (Supplementary Table 1). This method enables the
computation of the three-dimensional probability density func-
tion for the position of a dye molecule through geometric accessible
maltotriose (Supplementary Fig. 10a). These dissociation constants
were similar to those obtained from bulk measurements (Table 1).
Qualitatively, we obtained the same kinetic effects of the ligands
from the double mutant (A96W I329W) for maltose and maltotriose
(Fig. 4d,e and Supplementary Figs. 11 and 12), implying the gen-
erality of our observation. We also obtained similar dissociation
constants for the double mutant from smFRET analysis and bulk
measurements (Supplementary Fig. 10b and Table 1). For wild-
type MBP, we were not able to detect a transition between two sub-
states through smFRET in the presence of maltose (Supplementary
Fig. 13), mainly because the opening rate was too fast. In contrast,
in the presence of maltotriose, the transition between the two sub-
states was detectable, owing to a much longer dwell time in the

Figure 3 | Analysis of intrinsic dynamics a Cy3 Cy5 FRET d 0.6

of MBPs through smFRET. (a–c) The
Intensity

representative time traces of fluorescence 500

(AU)

Cy3 Cy5

partial closed form

0.4
npg

intensity (top) and FRET efficiency (bottom) 0

Ratio of

0.8
for wild-type MBP (a), the single mutant
FRET

0.4 0.2
(I329W) (b) and the double mutant (A96W
0
I329W) (c) in the absence of a ligand. The
0 2 4 6 8 10
decrease of FRET to 0 at around 9.7 s in Time (s) 0
a is mainly due to a bleaching of Cy5 dye.
SM

DM

Orange lines were added as a guide to make

1,000 0.08
the transitions clearer. Single and double b e
Intensity
(AU)

500
mutations were introduced in the hinge region,
closing rate (s )

0.06
–1

0
opposite the maltose-binding site of the MBP.
Intrinsic

0.8
(d) The relative population of the partially 0.04
FRET

0.4
closed state of the mutants in the absence 0
0.02
of maltose. The respective populations were 0 50 100 150
Time (s)
determined using the rates obtained from the 0
average dwell times for the open and partially
SM

DM

closed states. SM, single mutant; DM, double

mutant. (e,f) A comparison of the intrinsic c f 0.5
1,000
closing rates (e) and opening rates (f) of
Intensity
(AU)

500 0.4
opening rate (s )

the mutants in the absence of maltose. The

–1

0
intrinsic dynamics were analyzed from 48 and
Intrinsic

0.8 0.3
141 molecules showing a distinct transition
FRET

0.4
between two states among the single and 0.2
0
double mutants from three and four movies, 0 500 1,000 1,500 0.01
respectively. The error bars represent the s.d. Time (s) 0
of three or four data sets.
SM

DM

nature CHEMICAL BIOLOGY | vol 9 | MAY 2013 | www.nature.com/naturechemicalbiology 315

 article Nature chemical biology doi: 10.1038/nchembio.1213

Table 1 | Energetic and kinetic parameters for wild-type MBP and its variants
Energy of conformational Binding energy Dissociation constants Dissociation constants
Protein change («c, kcal mol−1) (D«, kcal mol−1) (Kd, nM)a (Kd, nM)b
Wild type 1.77c N.D. (maltose) N.D. 2,180 ± 190
–8.91 (maltotriose) 715 1,950 ± 260
Single mutant (I329W) 1.09 –9.48 (maltose) 99 48 ± 19
–9.82 (maltotriose) 56 53 ± 15
Double mutant (A96W I329W) 0.19 –10.39 (maltose) 7.2 4.7 ± 3.3
–10.19 (maltotriose) 10 2.3 ± 1.0
a
The dissociation constants were determined from a hyperbolic Hill fit of the relative population of a closed state calculated from the transition rates (Hill coefficient = 1.0) (Supplementary Fig. 10).
b
The dissociation constants were determined through ITC. Data represent the mean ± s.d. in triplicate experiments. cThe energy of conformational change for wild-type MBP was calculated on the
basis of data indicating that the probability of an intrinsic closed state is 5%. N.D., not determined.

closed state. The closing and opening dynamics analyzed for vary-
ing maltotriose concentrations (Supplementary Fig. 14) showed
a similar dependence of the ­binding and dissociation kinetics on
ligand concentration (Fig. 4f,g and Supplementary Fig. 15). We
were also able to obtain the dissociation constant of maltotriose for
wild-type MBP from the probability of the high FRET state (Online
Methods), and it was comparable to that obtained through bulk
measurement (Supplementary Fig. 10c and Table 1). Even though
the binding and dissociation kinetics could not be compared with
© 2013 Nature America, Inc. All rights reserved.
with binding in the partially closed form and dissociation in the
open form; and type IV (12%), with both binding and dissociation
in the partially closed form. Overall, more than 80% of binding and
dissociation events occurred in the open form, indicating a strong
preference for the open conformation. In wild-type MBP, the par-
tially closed form is much less populated than it is in the double
mutant, and a much stronger preference for the open conformation
is expected. The preference for the open conformation during bind-
ing seems to be due to the higher accessibility to the ligand.

the intrinsic closing and opening dynamics of the protein in this

case, we believe that the ligand recognition mechanism of the wild Ligand binding energy
type and the mutants are the same. The existence of a partially closed form itself has been interpreted
The analysis of the closing and opening kinetics of the MBPs as supporting evidence that this conformation has a critical role in
showed an acceleration of the closing rate with an increase in the the molecular recognition process4,29. However, our rigorous kinetic
ligand concentration as compared to the intrinsic closing rate. This analysis indicated a limited contribution of a partially closed form to
result therefore strongly supports the idea that the ligands pref- the ligand binding process, raising an issue regarding the biological
erentially bind the open conformation, followed by a structural roles of protein dynamics. We expected that consideration of the
transition of the proteins into a closed form, directly proving the
induced-fit mechanism, which has been the textbook explanation
a b No ligand c
for the molecular recognition process for the past 50 years. It is Cy3 Cy5 FRET Maltose
0.8

Opening rate (s–1)

Closing rate (s )
Maltotriose
Intensity

noteworthy that the MBP recognizes a ligand through an induced- 0.5

–1
(AU)

500 0.4 Sucrose 0.6

fit mechanism and not through the generally proposed selection 0
0.8
0.3 0.4
0.2
mechanism for proteins with conformational dynamics.
FRET

0.4 0.1 0.2

0 0 0
0 50 100 150
0
20
40
60
100
0

0
20
40
60
100
0
Analysis of ligand binding by three-color smFRET
8

8
Time (s) Ligand (nM) Ligand (nM)
To directly quantify the binding preference of a ligand to an open d e
conformation, we devised the use of three-color smFRET mea- 0.03 0.015

Opening rate (s–1)

Closing rate (s )
Intensity

–1
(AU)

500
surements27 using Cy7-labeled maltose (Supplementary Note 1).
npg

0.02 0.010
0
In three-color smFRET, the opening and closing dynamics of 0.8 0.01 0.005
FRET

0.4
MBP are monitored through Cy3-Cy5 FRET or Cy5 intensity, and 0 0 0
binding of maltose is monitored through Cy7 intensity. Owing to 0 50 100
Time (s)
150 0 2 4 6 8 10
Ligand (nM)
0 2 4 6 8 10
Ligand (nM)
steric hindrance by the linker used for dye labeling, we expected
f g
a ­considerable reduction in the binding affinity of Cy7-maltose 12
Opening rate (s–1)
Closing rate (s )
–1

6 10
Intensity

compared to that of unlabeled maltose. Nonetheless, we were able 700

(AU)

8
0 4 6
to observe distinct binding events (jumps of Cy7 signal and con- 0.8 2 4
FRET

0.4
current drops of Cy5 signal) of Cy7-maltose to the double mutant, 0 0
2
0
A96W I329W, at high ligand concentrations (Fig. 5a). Binding 0 50 100 150
200
400
600
8 0
1,000
00
200
40 0
600
8 0
1,000
00

events were very short, suggesting that the modification on malt-
ose was a hindrance. The increase in Cy7 intensity indicates the
binding of Cy7-maltose to the MBP, and the subsequent decrease
reflects the release of the ligand from the MBP. It was noteworthy
that the binding and dissociation events of Cy7-maltose occurred
in both the open and closed conformations of MBPs but happened
more frequently in the open conformation (Fig. 5a). To measure the
preference of the ligand binding and dissociation events for the
open and the partially closed forms, we made a correlation plot of
Cy3-Cy5 FRET efficiencies both at the binding and dissociation
points for the binding events of single Cy7-maltose (Fig. 5b). We
could classify single binding and dissociation events into four
categories: type I (75% of 479 events), with both binding and the
dissociation in the open form; type II (6%), showing binding in the
open form and dissociation in the partially closed form; type III (7%),
Time (s)
Ligand (nM) Ligand (nM)
Figure 4 | Kinetic analysis of conformational dynamics for MBPs in the
presence of a ligand. (a) Representative time traces of the fluorescence
intensity and FRET efficiency for the single mutant (I329W) at varying
maltose concentrations: top, no maltose; middle, 20 nM; bottom, 100 nM.
Orange lines were added as a guide to make the transitions clearer.
(b,c) Closing rates (b) and opening rates (c) as a function of ligand
concentration for the single mutant (I329W). (d,e) Closing rates (d) and
opening rates (e) as a function of ligand concentration for the double mutant
(A96W I329W). (f,g) Closing rates (f) and opening rates (g) as a function
of ligand concentration for wild-type MBP. In the latter case, a ligand-free,
partially closed state was not directly detectable through smFRET, and the
closing rate was set to 0 for the fitting. The error bars represent the s.d.
All data sets used in this analysis are represented in Supplementary Table 2.

316 nature chemical biology | vol 9 | MAY 2013 | www.nature.com/naturechemicalbiology

 Nature chemical biology doi: 10.1038/nchembio.1213
Figure 5 | Simultaneous analysis of Cy7-maltose a Cy5 signal
binding and conformational dynamics of the double
mutant through three-color smFRET. 0.8
Normalized intensity
0.4
(a) Representative intensity time traces of Cy5
0
fluorescence (red, top) and Cy7 fluorescence (gray,
0.8
bottom) normalized to the sum of Cy3, Cy5 and Cy7
0.4
fluorescence intensities in the presence of 400 nM 0
Cy7-maltose under excitation at 532 nm. Yellow- 0 100
shaded regions represent the partially closed states
of the double mutant (A96W I329W). The binding
of Cy7-maltose to an open state and a partially closed state are denoted by the open and solid arrowheads, respectively. (b) The relative distribution of
the FRET states before the binding and after the dissociation of Cy7-maltose from 479 binding and dissociation events of 52 molecules. Each data point is
represented through a gray-scaled two-dimensional Gaussian distribution. The shades of gray represent the relative density of the data points or events.
Red dashed lines were added for a clearer visualization of the four species (type I, binding to an open form followed by dissociation from the same state;
type II, binding to an open form and dissociation from a partially closed form; type III, binding to a partially closed form and dissociation from an open form;
and type IV, binding to a partially closed form followed by dissociation from the same state).
energetics of ligand binding for the three MBP variants would ­provide
some insight. Because ligand binding is associated with structural
changes in the MBPs, the dissociation constant of the ligand reflects
both the binding energy and the energy cost required for the con-
formational transition. When the binding energy is limited, as is
usually observed during small-ligand recognition processes, and if
© 2013 Nature America, Inc. All rights reserved.
the energy cost required for the structural transition is too large,
physiologically relevant binding affinities cannot be achieved.
Therefore, we determined both the conformational energy costs
for the closing transition and the ligand binding energies for the
wild type and single and double mutants (Online Methods). The
correlation between the structural energy costs and the dissocia-
tion constants observed in the three MBP variants supports this
hypothesis (Table 1). It is more likely that the intrinsic dynamics
of MBP facilitate a large conformational change that occurs upon
sugar binding, which can be achieved with less energy expenditure.
This view is consistent with recent studies suggesting that confor-
mational dynamics may have a role in facilitating the transition
to a holo structure18,22 or in influencing the chemical step during
enzyme catalysis30,31.
DISCUSSION
Our approach using smFRET measurements enabled a kinetic
analysis that interconnects the ligand binding and intrinsic con-
formational dynamics in the protein, providing direct evidence
npg
demonstrating the molecular mechanism of MBP that drives ligand
recognition. Our results showed that the binding of a ligand to the
MBP proceeds through the induced-fit mechanism and not through
the generally proposed conformational selection mechanism,
although different conformational substates exist. Our study also
revealed that the question of whether the recognition of the interact-
ing molecules is ‘induced’ or ‘selected’ is a problem of kinetics rather
than of thermo­dynamics, which deals with the relative probabilities
of the conformational substates. In addition, the mere presence of
conformational dynamics was insufficient evidence to indicate the
molecular recognition mechanism. An important question to be
answered is whether the ligand binds both open and partially closed
conformations of the MBP. Our three-color smFRET experiments
using Cy7-labeled maltose as a ligand showed that Cy7-maltose can
bind both open and partially closed conformations of the double
mutant and shows a preference for the open conformation, although
a considerable reduction in the binding affinity of Cy7-maltose was
expected, owing to the steric hindrance by the linker used for dye
labeling. We therefore expect that intact ligands can bind both con-
formations of an apo wild-type MBP but prefer an open conforma-
tion. Because the ligand binds both substates of the MBP, followed
by a conformational change, the results of three-color smFRET
measurements also indicate that the molecular recognition of MBP
follows an induced-fit mechanism.
nature CHEMICAL BIOLOGY | vol 9 | MAY 2013 | www.nature.com/naturechemicalbiology
article
Cy7 signal b
0.7
IV: 12%
(post-bound state)
II: 6%
FRET efficiency
0.6
0.5
III: 7%
0.4
I: 75%
200 300 0.4 0.5 0.6 0.7
Time (s) FRET efficiency
(pre-bound state)
Recent studies with NMR dispersion relaxation and spectro-
scopic techniques have revealed the existence of the conformational
substates of proteins29,32–35 and have suggested the necessary roles
of the intrinsic dynamics of the protein in molecular recognition,
leading to a development of dynamics-coupled recognition mech-
anisms. Much effort has been made to understand the interplay
between protein dynamics and molecular recognition. In particular,
MBP, a member of the bacterial periplasmic-binding protein (PBP)
family, has been the subject of extensive studies owing to its large-
scale domain rearrangement and the existence of conformational
substates and exemplifies the controversy regarding the recognition
mechanism22,23. A large conformational change in an MBP by ligand
binding is considered a classic example of the induced-fit mecha-
nism. The dynamic structural transitions of MBP between a major
open (~95%) form and minor partially closed (~5%) form suggest
that the initial binding of a ligand through conformational selection
is followed by a conformational adjustment4. Meanwhile, simulation
studies have indicated that the induced-fit mechanism mainly
­governs the binding of a ligand to MBP, but both conformational
selection and induced-fit mechanisms have a major role in molecu-
lar recognition23. Similarly, two possible pathways were proposed for
MBP on the basis of accelerated molecular dynamics simulations36:
either an induced-fit model or a model involving both induced-
fit and conformational selection mechanisms. Thus, to dissect the
fundamental aspects of the recognition mechanisms, the intrinsic
dynamics and structural changes of the protein should be traced
over the entire course of the ligand binding process. Our kinetic
analysis of conformational dynamics in the absence and presence of
increasing ligand concentration provided direct evidence to dissect
the molecular recognition mechanism of MBP. The results of this
study indicated an induced-fit mechanism for MBP, but one that
differs from Koshland’s pure induced-fit model, where the protein is
considered to exist in a single, stable conformation. Taken together
with the three-color smFRET results, we propose an ‘extended
induced fit’ model in which the protein exists in the ensemble of
conformations and the ligands bind the existing substates, inducing
a structural transition to a ligand-bound, closed form. Our model
can be distinguished from the extended conformational selection
model, in which the ligands bind only a weakly populated, higher-
energy ‘bound-like’ conformation followed by structural adjustment
to a complete ligand-bound form.
The conformational diversity of proteins has been suggested to be
important in the evolution of molecular recognition4. The binding of
a ligand to all conformations is likely to contribute to low ligand speci-
ficity of the protein, whereas the conformational selection mechanism
gives rise to narrow ligand specificity because generally a single sub-
state is engaged in the molecular recognition process. Considering
MBP’s role in E. coli and its broad specificity for sugars, its molecular
recognition mechanism will be advantageous for the survival of
317
 article
microorganisms on diverse sugars. The presence of different substates
in an MBP may allow an interaction with more diverse types of sug-
ars. It has been suggested that less specific PBPs, such as glutamine-
binding protein (GlnBP), are unlikely to have a partially closed con-
formation37, whereas PBPs showing broad specificity, such as lysine-,
arginine-, ornithine-binding (LAO) protein38; ribose-binding protein
(RBP)39; glucose/galactose-binding protein (GGBP)40; and choline-
binding protein (ChoX)41 seem to have a partially closed substate.
Molecular recognitions are central to all biological processes. Both
the induced-fit and conformational selection mechanisms are likely to
have crucial roles in molecular recognition. The approach presented
in this study will provide a new paradigm in elucidating the molec-
ular mechanisms by which other proteins showing conformational
dynamics recognize and bind their cognate ligands, assisting in the
design of more potent drugs and even proteins with new functions.
Received 6 August 2012; accepted 21 February 2013;
published online 17 March 2013
Methods
Methods and any associated references are available in the online
version of the paper.
© 2013 Nature America, Inc. All rights reserved.
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Acknowledgments
This work was supported by the Pioneer Research Program for Converging
Technology (2008-2000218), Advanced Biomass R&D Center (2011-0031363),
the Intelligent Synthetic Biology Center (2011-0031950) and the Brain Korea 21
program of the Ministry of Education, Science and Technology (H.-S.K.) and Creative
Research Initiatives (2009-0081562) and the World-Class University program of the
National Research Foundation of Korea (S.H.). We thank S. Mowbray and D.D. Boehr
for helpful discussions.
Author contributions
E.K. and J.M.C. prepared and characterized the MBP variants. E.K. and S.L. designed,
performed and analyzed the single-molecule experiments. A.J and H.-S.L prepared and
characterized the Cy7-maltose. E.K., S.L., S.H. and H.-S.K. interpreted the data and wrote
the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Additional information
Supplementary information and chemical compound information is available in the
online version of the paper. Reprints and permissions information is available online at
http://www.nature.com/reprints/index.html. Correspondence and requests for materials
should be addressed to S.H. or H.-S.K.
nature chemical biology | vol 9 | MAY 2013 | www.nature.com/naturechemicalbiology
 ONLINE METHODS
Reagents. Sugars (maltose, maltotriose and sucrose) used as ligands were
purchased from Sigma. Cy3-maleimide, Cy5-maleimide and Cy7-mono
NHS ester fluorophores were purchased from GE Healthcare. Cy7-labeled
maltose (10) was obtained through conjugation between the poly(ethylene
glycol)-amine linked maltose (9, Medigen, Korea) and Cy7-mono NHS ester
(GE Healthcare). Details on their synthesis and preparation are described in
Supplementary Note 1.
Protein expression and purification. MBP from pMAL-c2x (New England
Biolabs) was linked with a His6 tag and the biotin acceptor peptide for
biotinylation at the N terminus. The resulting gene was cloned into the pET21a
vector (Novagen). Single and double mutants of MBP were constructed by
introducing one (I329W) and two (A96W I329W) mutations, respectively,
using site-directed mutagenesis. For dye labeling, two cysteine residues were
introduced in the N-terminal (K34C) and the C-terminal (R354C) domains of
the wild type and mutants. MBP variants were expressed in E. coli BL21(DE3)
cells harboring pProTrc-BirA, which contains the biotin ligase gene from
pBIRAcm (Avidity). Proteins were purified by immobilized metal affinity
chromatography using Ni-NTA superflow matrix (Qiagen). The purified
MBP was further desalted with PBS (pH 7.2) using a PD-10 desalting column
(GE Healthcare) for dye labeling. Protein concentration was measured by the
Bradford method. HABA/Avidin reagent (Sigma) was used to quantify the
amount of biotinylated protein.
© 2013 Nature America, Inc. All rights reserved.
Dye labeling. MBP variants containing two cysteine residues were labeled
with Cy3- and Cy5-maleimide dyes (GE Healthcare). After reducing the sur-
face exposed–cysteine residues with 100-fold excess of Tris(2-carboxyethyl)
phosphine (TCEP) hydrochloride for 10 min, proteins were incubated with a
ten-fold molar excess of the two dyes in anhydrous dimethylformamide (DMF)
in equimolar ratio for 2 h at room temperature. Unreacted dyes were removed
from the protein solution by desalting with a PD-10 column (GE Healthcare)
using TNG buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 5% glycerol). The
labeling efficiency was determined by measuring the protein concentration
(absorbance at 280 nm) and the dye concentrations (absorbance at 552 nm and
at 650 nm for Cy3 and Cy5, respectively).
CD analysis. CD measurements were conducted before and after dye labeling
for wild-type MBP and its variants using a JASCO J-815 (JASCO) with a quartz
cell of path length 0.01 cm at 25.0 °C. Protein solutions were prepared in
sample buffer (10 mM Tris-Cl, 50 mM NaCl, 5% glycerol, pH 7.5), and the
protein concentration ranged from 0.1 mg/ml to 0.5 mg/ml. CD spectra were
taken between 200 nm and 260 nm with 1-nm increments, and the background
spectrum of the buffer solution was subtracted.
npg
Assay of maltose-binding activity. Maltose-binding activities of MBP variants
were tested by the amylose-binding assay using the MBP Excellose Spin Kit
(TaKaRa Bio). Purified proteins were added on to a MBP Excellose spin column,
incubated at room temperature for 2 min and centrifuged at 700g for 2 min.
Following washing and elution using binding buffer (50 mM Tris, pH 7.0,
100 mM NaCl) and elution buffer (binding buffer containing 10 mM maltose),
all filtrates were subjected to SDS-PAGE (12%) analysis.
ITC. All calorimetric titrations were performed using the iTC-200
Microcalorimeter (Microcal) at 7.0 °C. The protein concentration in the calo-
rimeter cell ranged from 0.02 mM to 0.2 mM, and the ligand concentration in
the injector varied from 0.2 mM to 2 mM such that the molar ratio of ligand to
macromolecule, following the last injection, was approximately 2. Protein and
ligand solutions were prepared in sample buffer (50 mM NaPO4, 100 mM NaCl,
pH 7.0). Data were analyzed using the software provided by the supplier.
smFRET experiments. Cleaned quartz slides and coverslips were coated with
polyethylene glycol and biotinylated polyethylene glycol (Laysan Bio) to pre-
vent nonspecific adsorption of proteins to the surface42. A sample chamber
was created between a cleaned quartz slide and a coverslip using double-sided
adhesive tape. Proteins were immobilized on the surface by the successive
addition of streptavidin (Invitrogen) and 0.5–1.5 nM biotinylated protein in
TN buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl). To reduce photobleach-
ing and blinking of dyes, a protocatechuic acid (PCA)/protocatechuate-3,4-
dioxygenase (PCD) oxygen-scavenging system43 and the blinking suppressant
doi:10.1038/nchembio.1213
Trolox were used42. Single-molecule fluorescence images were taken in imaging
buffer (10 mM Tris-HCl, pH 8.0, 50 nM PCD, 2.5 mM PCA, 1% (v/v) Trolox)
using a home-built prism-type total internal reflection fluorescence micro-
scope. Three-color smFRET experiments were performed using Cy7-labeled
maltose. Cy3 was excited by a 532-nm laser (Compass215M; Coherent) for
two-color FRET, and the alternative excitation of Cy3 and Cy5 was performed
by a 532-nm and 633-nm laser (LGK 7626S; Lasos) using a mechanical shut-
ter for three-color smFRET27. The fluorescence signals of the three dyes were
collected using a water immersion objective lens (UPlanSApo 60×; Olympus)
and filtered through a 540-nm long-pass filter (LP03-532RU-25; Semrock) and
a 633-nm notch filter (NF03-633E-25; Semrock) to reject scattered excitation
laser lines. The emissions were then separated by dichroic mirrors (635dcxr;
Chroma Technology) and detected by an electron-multiplying charged-
coupled-device (Ixon DV897; Andor).
Analysis of smFRET data. To collect only the dual-labeled proteins from
heterogeneous populations, a few hundred single-molecule spots from the
images of an acceptor detection channel were picked following 532-nm
laser excitation. Time traces of Cy3 and Cy5 signals from a single molecule
were extracted from a recorded movie file using IDL software (ITT Visual
Information Solutions) and analyzed using custom software written in
MATLAB (MathWorks). The donor (ID) and acceptor (IA) fluorescence inten-
sities from each single molecule were measured, and the FRET efficiencies
(EFRET) were calculated using the intensity-based equation; IA / (IA + ID)42. After
background subtraction and cross-talk correction, we selected real molecule
traces showing anticorrelations between the donor and acceptor or single-step
photobleaching. To obtain the kinetic times from the time traces, FRET effi-
ciency levels were identified as orange lines within all figures by the standard
threshold method. The dwell times were obtained in the absence and presence
of varying ligand concentrations by fitting dwell-time histograms of each state
to a single-exponential decay curve42, and the transition rates were determined
by inverting the dwell times.
Determination of dissociation constant. The reaction observed through the
smFRET assay is depicted as follows.
k
f

O C (1)
kb
where kf is the transition rate from an open form (O) to a closed form (C),
and kb is the transition rate from a closed form to an open form. In the case of
the single (I329W) and double (A96W I329W) mutants, kf and kb at varying
ligand (L) concentrations are given by
kf = kf0 + a ⋅[L]
kb = kb0 (2)
Our empirical result for the closing rate is consistent with a picture that a
ligand forms an encounter complex with the open form and subsequently
induces a conformational transition to the closed form44:
r [ L] r
b

O c→C ⋅ L
 O ⋅ L 
 (3)
ru
where rb, ru and rc are binding, unbinding and closing rate constants, respec-
tively. In this scheme,
rbrc
a=
ru
At equilibrium, therefore,
[C] kfo + a ⋅[L]
(kf0 + a ⋅[L]) ⋅[O] = kbo ⋅[C] → = (4)
[O] kbo
From equation (4), the probability of a closed state (Pclosed) is expressed as
follows:
[C] k o + a ⋅[L]
Pclosed = = o f o (5)
[O]+[C] kb + kf + a ⋅[L]
nature CHEMICAL BIOLOGY
 which can be modified as follows: The chemical potential of an ideal solution is generally written as
o o [L] (6) 1 [L] 1
Pclosed = Pclosed + (1 − Pclosed )⋅ m = mo + ln = mo + ln[L]
K d + [L] b [L]o b (10)

where μo is the chemical potential of the reference state ([L]o = 1 M).

where
By substituting equation (10) into equation (8) and (9), we obtain
o kfo
Pclosed =
(kbo + kfo )
− b  e b − mo − 1 ln[ L]+ e c 
b
e − be c + e   e − be c + [L] ⋅ e − b ( Δe +e c )
Pclosed = =
is the probability of a partially closed state in the absence of a ligand, and − b  e b − mo − 1 ln[ L]+ e c 
b
1 + e − be c + [L] ⋅ e − b ( Δe +e c )
1 + e − be c + e  

kbo + kfo
Kd = e b Δe + [L] e b Δe + [L]
(a ) = =
e b ( Δe + e c )+ b Δe
e + [L] e b Δe
⋅ (1 + e be c ) + [L] (11)
is the dissociation constant of a ligand. The dissociation constants
for the single and double mutants were obtained by fitting the closed
state probabilities at varying ligand concentrations to equation (6). − b  e b − mo − 1 ln[ L]+ e c 
b
e   [L] ⋅ e − b ( Δe +e c )
In the case of wild-type MBP, a ligand-free partially closed state was Pbound = = − be c
not directly detectable through smFRET, and Pclosed
 was set to 0 for − b  e b − mo − 1 ln[ L]+ e c 
b
1+ e + [L] ⋅ e − b ( Δe +e c )
1 + e − be c + e  
the fitting.
[L] [L]
Calculation of the binding and conformational energies. On the basis of = =
e b ( Δe +e c )+ e b Δe + [L] e b Δe ⋅ (1 + e be c ) + [L] (12)
the concept that dissociation constant (Kd) reflects the binding energy and
the conformational change energy, we calculated respective energy values
© 2013 Nature America, Inc. All rights reserved.

where we introduced the notation for the binding free energy (the energy
from the dissociation constant (Kd) and the probability of an intrinsic
 difference upon taking a ligand from solution and placing it on the protein),
closed state, Pclosed . In statistical mechanics, the relative probability of a state
Δε=εb−μo. Using equation (7), Pclosed can be rewritten as follows.
is determined by a Boltzmann factor of the state, e−βε, where β is 1/(kBT),
and ε is the free energy of the state45. In the absence of a ligand, there exist two
o e b Δe + [L] 1
distinct states: an open state and a partially closed state. The probability of a Pclosed − Pclosed = −
partially closed state ( Pclosed
 ) is given by e b Δe
⋅ (1 + e be c ) + [L] 1 + e be c
(13)
e be c [L]
− be c = be c
⋅ b Δe be c
o e 1 (7) 1+ e e ⋅ (1 + e ) + [L]
Pclosed = =
1 + e − be c 1 + e be c
o o [L] (14)
where εc is the free energy of the partially closed state relative to the open → Pclosed = Pclosed + (1 − Pclosed )⋅
e b Δe ⋅ (1 + e be c ) + [L]
state. Therefore, the conformational energy (εc, the energy required for the

transition form an open state to a partially closed state) is obtained from Pclosed . By comparing equation (14) with equation (6), we obtain the following

Pclosed was directly obtained from smFRET experiments for the single and equation:
double mutants, whereas a reported value (5%) was used for wild-type
MBP. In contrast, there exist three states in the presence of a ligand: (i) a K d = e b Δe ⋅ (1 + e be c ) (15)
ligand-free open state (ε=0), (ii) a ligand-free partially closed state (ε=εc) and
(iii) a ligand-bound closed state (ε=εb−μ+εc, where εb and μ are the ligand- which allows us to determine the binding free energy (Δε) from the dissocia-
receptor interaction energy and the chemical potential of the ligand, respectively; tion constant (Kd). For wild-type MBP, the same relation between Kd and Δε
(equation (15)) is obtained from equation (12).
npg

we assumed that the conformational energy difference between a ligand-free

partially closed state and a ligand-bound closed state was negligible). Therefore,
the probabilities in either a partially closed state or a closed state (Pclosed) and
the probability in a ligand-bound closed state (Pbound ) are given by
e − be c + e − b (e b − m +e c )
Pclosed =
1 + e − be c + e − b (e b − m +e c )
e − b (e b − m + e c )
Pbound =
1 + e − be c + e − b (e b − m +e c )
42. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET.
Nat. Methods 5, 507–516 (2008).
43. Aitken, C.E., Marshall, R.A. & Puglisi, J.D. An oxygen scavenging system for
improvement of dye stability in single-molecule fluorescence experiments.
(8) Biophys. J. 94, 1826–1835 (2008).
44. Weikl, T.R. & von Deuster, C. Selected-fit versus induced-fit protein binding:
kinetic differences and mutational analysis. Proteins 75, 104–110 (2009).
45. Phillips, R., Kondev, J. & Theriot, J. Physical Biology of the Cell.
(9)
Ch. 6 (Garland Science, New York, 2009).

nature chemical biology doi:10.1038/nchembio.1213