Conformational modulation and hydrodynamic radii of
CP12 protein and its complexes probed by fluorescence
correlation spectroscopy
Rigneault1,
Satish Babu Moparthi1, Gabriel Thieulin-Pardo2, Pascal Mansuelle3, Herve
2
1
ro
^ me Wenger
Brigitte Gontero and Je
, France
1 Centrale Marseille, Institut Fresnel, Aix Marseille Universite
nerge
tique et Inge
nierie des Prote
ines, Aix Marseille Universite
, France
2 Laboratoire de Bioe
omique, Marseille Prote
omique, Institut de Microbiologie de la Me
diterrane
e, France
3 Plate-forme Prote
Keywords
CP12; fluorescence correlation
spectroscopy; GAPDH; hydrodynamic
radius; intrinsic disorder proteins; PRK
Correspondence
S. B. Moparthi, CNRS, Centrale Marseille,
,
Institut Fresnel, Aix Marseille Universite
UMR 7249, 13013 Marseille, France
Fax: +(33) 4 9 128 80 67
Tel: +(33) 4 9 128 84 94
E-mail: [email protected]
(Received 7 February 2014, revised 30 April
2014, accepted 16 May 2014)
doi:10.1111/febs.12854
Light/dark regulation of the Calvin cycle in oxygenic photosynthetic organisms involves the formation and dissociation of supramolecular complexes
between CP12, a nuclear-encoded chloroplast protein, and the two enzymes
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) and
phosphoribulokinase (PRK) (EC 2.7.1.19). Despite the high importance of
understanding the structural basis of the interaction of CP12 with GAPDH
and PRK to investigate the regulation of the Calvin cycle, information is still
lacking about the structural remodulation of CP12 and its complex formation. Here, we characterize the diffusion dynamics and hydrodynamic radii of
CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK
using fluorescence correlation spectroscopy experiments. We quantify a
hydrodynamic radius of 3.4 0.2 nm for the CP12 protein with an increase
up to 5.2 0.3 nm upon complex formation with GAPDH and PRK. In
addition, unfolding experiments reveal a 1.6- and 2.0-fold increase respectively
of the hydrodynamic radii for the N-terminal and C-terminal cysteine CP12
mutant proteins compared with their native folded structures. The different
behavior of the CP12 mutant proteins during hydrophobic collapse transition
is a direct clue to different structural orientations of the CP12 mutant proteins. These different structures are expected to facilitate the binding of either
GAPDH or PRK during binary complex and ternary complex formation.
Structured digital abstract
GAPDH, CP12 and PRK physically interact by fluorescence correlation spectroscopy (View interaction)
CP12 and PRK bind by fluorescence correlation spectroscopy (View interaction)
GAPDH and CP12 bind by fluorescence correlation spectroscopy(View interaction)
Introduction
CP12 is a small chloroplast protein present in many
photosynthetic organisms including the eukaryotic
unicellular green alga Chlamydomonas reinhardtii,
where it is composed of 80 amino acids and has a
molecular mass of 8.5 kDa [1–6]. CP12 plays a key
role in regulating the Calvin cycle by forming supramolecular complexes with glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and phosphoribulokinase
(PRK) downregulating their activity [3,7–13]. In the
dark, oxidized CP12 forms a supramolecular complex
Abbreviations
DTT, dithiothreitol; FCS, fluorescence correlation spectroscopy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GuHCl, guanidinium
hydrochloride; IDP, intrinsically disordered protein; PRK, phosphoribulokinase.
3206
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
S. B. Moparthi et al.
with GAPDH and PRK, while in the light, due to the
action of reduced thioredoxins and consequent disruption of disulfide bridges, the supramolecular complex
dissociates [5,14]. Understanding the structural basis of
the interaction of CP12 with GAPDH and PRK is
thus of high importance to investigate the regulation
of the Calvin cycle.
CP12 is an intrinsically disordered protein (IDP)
[2,5,6,15]. IDPs are proteins that under physiological
conditions lack a rigid well-folded structure and exhibit low compactness and high flexibility. Their
sequences are enriched in polar and charged residues
and they have low content of hydrophobic amino acids
that normally form the core of folded globular proteins [16–18]. IDPs are mainly found in eukaryotes
(30–50% of eukaryotic proteins) and only a small proportion of these proteins are of eubacterial or archaeal
origin (less than 5%) [19–21]. In plants only a few
examples of IDPs have been described beside CP12,
including dehydrins [22,23] and an Mn stabilizing protein belonging to photosystem II [24].
Several ensemble-based methods have been used to
investigate the role and above all the interaction of
CP12 with GAPDH and PRK, including size exclusion
chromatography, immunoprecipitation assays, nuclear
magnetic resonance or electron paramagnetic resonance spectroscopy [2,10,25,26]. These indicate that
GAPDH and PRK proteins bind to different sites on
CP12 [7,8,10]. CP12 from C. reinhardtii bears four cysteine residues at positions 23, 31, 66 and 75, which
form two consecutive disulfide bridges at both N-terminal (Cys23-Cys31) and C-terminal (Cys66-Cys75)
extremities of the protein (Fig. 1A) [1,2,4–6,27]. The
N-terminal disulfide bridge is expected to be involved
in the binding of PRK homo-dimer, while the C-terminal disulfide bridge appears necessary for redox regulation of the GAPDH homo-tetramer [1,2,4–6,27]
(Fig. 1B).
Here, we take advantage of fluorescence correlation
spectroscopy (FCS) experiments to probe the diffusion
dynamics of CP12 molecules from C. reinhardtii upon
binding to GAPDH and PRK. FCS is a powerful and
versatile method based on the statistical analysis of the
temporal fluctuations affecting the fluorescence intensity from a few diffusing molecules [28–37]. It allows
the investigation of a large variety of dynamic processes
and photophysical properties, including translational
diffusion, molecular concentrations, fluorescence
brightness, chemical kinetics and binding reactions.
The aim of this work was to determine the hydrodynamic radii of the wild-type CP12 (CP12wt) and its
two site-specific mutant proteins altered both at the Nterminus (cysteine residue at position 31 replaced by a
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
CP12 structural modulation
serine, referred to as CP12C31S) and at the C-terminus
(cysteine residue at position 75 replaced by a serine,
referred to as CP12C75S). To investigate the binding
properties between CP12, GAPDH and PRK, we
monitor the changes of the hydrodynamic radii upon
interaction with PRK and GAPDH. Our results clearly
point out the crucial influence of the mutations affecting the disulfide bridges for the complex formation
between CP12, GAPDH and PRK. Moreover, the
change in hydrodynamic radii of the CP12 mutant
proteins upon denaturation with increasing concentration of guanidinium hydrochloride (GuHCl) is analyzed using FCS. The different behavior of the CP12
mutant proteins during hydrophobic collapse transition provides a direct clue to different structural orientations resulting from the mutation of a single
disulfide bridge.
Results
CP12wt interaction with GAPDH and PRK
FCS results for CP12wt are summarized in Fig. 2 and
Table 1. The measured hydrodynamic radius of
at pH 8.0, and is similar to
CP12wt is around 34 A
the hydrodynamic radii found for the two mutants
CP12C31S and CP12C75S. In addition, longer diffusion times were obtained with CP12wt both in the
presence of GAPDH or PRK alone and also in the
presence of the two enzymes simultaneously. The presence of GAPDH significantly increased the hydro (Fig. 2B). Similarly,
dynamic radius from 34 to 47 A
the addition of PRK alone increased the apparent
Forhydrodynamic radius of CP12wt from 34 to 43 A.
mation of the whole complex CP12wt–GAPDH–PRK
showed further significant increase of the apparent
Upon treatment with
hydrodynamic radius up to 52 A.
dithiothreitol (DTT) as reducing agent, all hydrodynamic radii were similar to that of CP12wt alone and in
This set of experiments indicates
the range 34–36 A.
that no complex was formed between CP12wt, GAPDH
and/or PRK in the presence of DTT (Fig. 2C).
CP12 self-association
We checked that under the experimental conditions
the CP12 mutant proteins were in the form of
monomers and did not tend to form aggregates. To
this end, the fluorescence correlation functions were
measured by keeping the labeled CP12 concentration
constant at 40 nM and adding increasing concentrations of unlabeled CP12 from 500 nM up to 50 lM.
Regardless of the CP12 concentration, the hydrody-
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CP12 structural modulation
S. B. Moparthi et al.
A
C
C23
C31
kDa 130
100
70
50
1
MW
2
3
4
5
35
25
C75
15
C66
N
1)
2)
3)
4)
5)
C
B
GAPDH
PRK
CP12 wild-type
CP12 C31S
CP12 C75S
N
C
N
C
PRK-CP12C75S
GAPDH-CP12C31S
CP12
C
N
PRK
GAPDH
C
N
GAPDH-CP12-PRK
(Fig. 3A). These results
namic radius was about 32 A
indicate that in vitro there is no substantial interaction
between the labeled and non-labeled CP12 protein
even at concentrations up to 50 lM and that, very
likely, the CP12 exists as a monomer in the absence of
GAPDH and PRK.
GAPDH–PRK–CP12 mutant interactions
The role of the disulfide bridges of CP12 in the interaction with GAPDH and PRK can be investigated
through the use of CP12 mutant proteins affected
3208
Fig. 1. Structure and interaction of CP12
with GAPDH and PRK. (A) 3D structural
model of Chlamydomonas reinhardtii
CP12. (B) Schematic representation of the
association–dissociation of GAPDH, CP12
and PRK proteins. Both in vivo or in vitro
CP12 forms two disulfide bridges, one at
the N-terminus and one at the C-terminus,
which then act as linkers for the formation
of non-covalent complexes GAPDH–CP12,
PRK–CP12 and GAPDH–CP12–PRK. (C)
SDS gel (4–10%) stained with Coomassie
Blue to test the homogeneity of GAPDH
(lane 1), PRK (lane 2), CP12wt (lane 3),
CP12C31S (lane 4) and CP12C75S (lane 5).
either at the N-terminal disulfide bridge (CP12C31S)
or at the C-terminal disulfide bridge (CP12C75S). Figure 3B displays normalized FCS correlation traces
obtained on the N-terminal disulfide bridge mutant
CP12C31S in the absence and presence of GAPDH or
PRK. In the presence of GAPDH, the correlation
traces shift towards longer diffusion times indicating
the formation of a sub-complex CP12C31S–GAPDH
(Fig. 3B). Meanwhile, the presence of PRK did not
induce any noticeable change in the correlation traces
of CP12C31S compared with the CP12 mutant alone.
The hydrodynamic radii deduced from the FCS data
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Normalised auto correlation g (t)
S. B. Moparthi et al.
CP12 structural modulation
Table 1. Diffusion time and hydrodynamic radii for CP12wt protein
in the presence and absence of GAPDH, PRK proteins and DTT.
A
1.0
CP12 wild-type
Sample
0.8
sd (ls)
RH (
A)
352
479
357
422
373
543
353
33.8
46.1
34.3
40.6
35.8
52.2
33.9
+ GAPDH
CP12wt
CP12wt
CP12wt
CP12wt
CP12wt
CP12wt
CP12wt
+ PRK
0.6
+ GAPDH + PRK
0.4
0.2
0.0
0.01
0.1
1
10
+
+
+
+
+
+
GAPDH
GAPDH + DTT
PRK
PRK + DTT
GAPDH + PRK
GAPDH + PRK + DTT
1.7
2.3
1.7
2.0
1.7
2.6
1.6
100
Time (ms)
B
Hydrodynamic radii (nm)
5.5
5.0
4.5
4.0
3.5
3.0
2.5
CP12Wt
C
+ GAPDH
+ PRK
Normalised auto
correlation g (t)
1.0
5.5
Hydrodynamic radii (nm)
+ PRK
+ GAPDH
5.0
4.5
0.8
0.6
0.4
0.2
0.0
0.01
0.1
4.0
1
10
100
Time (ms)
3.5
GAPDH binding site on CP12 unaffected.
Furthermore, successive additions of PRK to the
CP12C31S–GAPDH complex did not modify the
apparent hydrodynamic radius of CP12. This result
indicates that PRK is not able to bind to the
CP12C31S mutant even in the presence of GAPDH.
Results for the other CP12C75S mutant affected at
the C-terminal disulfide bridge are summarized in
Fig. 4A,B and Table 2. Both CP12 mutant proteins
CP12C31S and CP12C75S have a comparable hydro at pH 8.0. Unlike the correladynamic radius of 32 A
tion traces of CP12C31S, the traces obtained with
CP12C75S showed a shift towards longer diffusion
times in the presence of PRK, while the presence of
GAPDH did not significantly increase the diffusion
dynamics. Here, the addition of PRK increased the
apparent hydrodynamic radius of CP12C75S from 32
This indicates that the C-terminal C75S mutato 38 A.
tion on CP12 does not affect the binding site for PRK
while it significantly hampers the subsequent formation
of a complex with GAPDH.
3.0
2.5
CP12Wt
+ GAPDH
+ DTT
+ PRK
+ DTT
+ GAPDH
+ PRK
+ DTT
Fig. 2. FCS results on the wild-type CP12 protein. (A) Normalized
FCS correlation traces and (B) apparent hydrodynamic radii
obtained in the presence of GAPDH or PRK or both. Five-fold
molar excess proportions of GAPDH and PRK were used with
CP12wt, by keeping CP12wt constant at 10 nM. (C) Reduction
effect of the disulfide bridges by 1 mM DTT on the values of the
hydrodynamic radii of all samples.
in Fig. 3B are summarized in Fig. 3C and Table 2 for
the N-terminal mutant CP12C31S. The addition of
GAPDH increased the apparent hydrodynamic radius
while the addition of
of CP12C31S from 32 to 47 A
PRK did not affect the hydrodynamic radius. This
result indicates that the N-terminal C31S mutation on
CP12 severely affects PRK binding while leaving the
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Effect of PRK concentration on CP12–PRK
complex formation
To investigate the effect of PRK concentration on the
formation of the CP12C75S–PRK complex, the apparent hydrodynamic radius of CP12C75S at a constant
concentration of 100 nM was analyzed when increased
concentrations of unlabeled PRK protein up to a
molar excess of 1 : 20 were added (Fig. 4C). Concentrations of PRK in a ratio less than 1 : 1 did not
induce any significant change in the hydrodynamic
radius. At concentration ratios higher than 1 : 1 the
CP12C75S–PRK complex was formed affecting the
apparent hydrodynamic radius until saturation was
reached at a molar excess of 1 : 4 of PRK compared
with CP12. Our observations tend to indicate a cooperative binding, involving at least two conformers of
CP12 in equilibrium with different affinities for PRK.
Binding of PRK will displace the equilibrium towards
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CP12 structural modulation
S. B. Moparthi et al.
1.0
CP12C31S
Table 2. Diffusion time, hydrodynamic radii and molecular
brightness CRM for CP12 mutant proteins in the presence and
absence of GAPDH and PRK proteins.
0.8
+ GAPDH
Sample
sd (ls)
RH (
A)
0.6
+ PRK
CP12C31S
CP12C31S + GAPDH
CP12C31S + PRK
CP12C31S + GAPDH + PRK
CPCP12C75S
CP12C75S + GAPDH
CP12C75S + PRK
CP12C75S + GAPDH + PRK
290
423
286
407
285
280
342
365
32.4
47.2
31.9
45.4
31.8
31.2
38.2
40.7
Normalised auto correlation g (t)
A
+ GAPDH + PRK
0.4
0.2
0.0
0.01
0.1
1
Time (ms)
10
100
B
Hydrodynamic radii (nm)
5.0
4.5
4.0
3.5
3.0
2.5
CP12C31S
+ GAPDH
+ PRK
+ GAPDH
+ PRK
CP12C31S
+ 500 nM
+ 5 µM
+ 50 µM
Hydrodynamic radii (nm)
C
3.4
3.2
3.0
2.8
Fig. 3. FCS data on the N-terminal disulfide bridge mutant
CP12C31S. (A) CP12C31S diffusion time independence of
increasing concentrations of unlabeled CP12C31S. (B) Normalized
FCS correlation traces and (C) deduced hydrodynamic radii
obtained on CP12C31S in the absence and presence of GAPDH or
PRK. The molar ratio was set to 1 : 1 with each species at a fixed
concentration of 100 nM.
the higher affinity conformer giving rise to the sigmoid
curve obtained (Fig. 4C).
Unfolding of CP12 mutant proteins analyzed by
FCS
To determine the structural transitional dimensions of
CP12 mutant proteins, increased concentrations of
3210
1.6
2.3
1.5
2.2
1.3
1.4
1.9
2.0
GuHCl up to 5 M were added and the hydrodynamic
radii of the partially denatured states were determined
by FCS. In the presence of high concentrations of
GuHCl, both the solvent’s refractive index and viscosity are changed. Both effects were taken into account
by calibrating the ratio of the transversal waist to the
solvent’s viscosity for each GuHCl concentration
(Fig. 5). With that calibration, the measured hydrodynamic radii of both Alexa Fluor 647 and Atto647N do
not vary upon addition of GuHCl, confirming our
experimental method.
Figure 6A represents the correlation functions of
both CP12 mutant proteins in the absence or presence
of GuHCl; all the data were fitted to a free single-component diffusion model with a diffusion time of sd and
an average number N of molecules by using Eqn (1)
(later). The accuracy of the fits was established using
residual distribution analysis. Figure 6B depicts the
hydrodynamic radii of both CP12 mutant proteins in a
series of increasing GuHCl concentrations. While
under native conditions both CP12C31S and
CP12C75S had similar hydrodynamic radii, the hydrodynamic radius values obtained at 5 M GuHCl for
strongly denatured CP12C31S and CP12C75S revealed
an increase of 1.6- and 2.0-fold respectively compared
with their corresponding native structures. Moreover,
difference in hydrodynamic radius
the significant 10 A
between the two mutant proteins under denatured conditions indicates that the N- and C-terminal mutant
proteins differ in structure.
Discussion
CP12 is very flexible and largely devoid of secondary
structure; however, when disulfide bonds are present,
a-helix content and the overall degree of order of
CP12 is increased [2]. This protein is able to bind
metal ions such as copper and nickel in C. reinhardtii
[38,39] and calcium in higher plant [40]. CP12 also
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
S. B. Moparthi et al.
CP12 structural modulation
A
1.0
+ GAPDH
0.6
+ PRK
0.4
+ GAPDH + PRK
150
140
130
120
0
1
2
3
4
5
4
5
[GuHCl] (M)
0.0
0.1
1
10
100
B
Time (ms)
500
Waist (nm)
480
B
4.5
4.0
460
440
420
400
380
3.5
3.0
C
2.5
CP12C75S
+ GAPDH
+ PRK
+ GAPDH
+ PRK
C
4.5
Refractive Index
Hydrodynamic radii (nm)
160
0.2
0.01
Hydrodynamic radii (nm)
180
170
CP12C75S
0.8
td (µs)
Normalised auto correlation g (t)
A
0
1
0
1
2
3
[GuHCl] (M)
1.44
1.42
1.40
1.38
1.36
1.34
1.32
4.0
2
3
4
5
[GuHCl] (M)
3.5
3.0
4 6 8
10
2
4 6 8
100
2
4 6 8
1000
2
PRK concentration (nM)
Fig. 4. FCS data on the C-terminal disulfide bridge mutant
CP12C75S. (A) Normalized FCS correlation traces and (B) deduced
hydrodynamic radii obtained on CP12C75S in the absence and
presence of GAPDH or PRK. In (A) and (B), the concentrations of
CP12C75S, GAPDH and PRK were 100, 500 and 500 nM
respectively, corresponding to a 5-fold molar excess of GAPDH
and/or PRK relative to CP12C75S. (C) Dependence of the
CP12C75S hydrodynamic radius on the concentration of PRK. The
labeled CP12C75S concentration was kept constant at 100 nM.
plays a role in oxidative stress as shown by antisense
CP12 mutant plants [41]. In early studies, other proteins such as malate dehydrogenase, elongation factor
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Fig. 5. Calibration procedure for the influence of increased
concentrations of GuHCl. (A) Diffusion time measured for Alexa
Fluor 647 as function of GuHCl concentration. (B) Effective
transversal waist deduced from the measurements in (A) and the
calibrated 0.7 nm hydrodynamic radius of Alexa Fluor 647 at 22 °C.
(C) The evolution of the transversal waist in (B) is found to
correlate with increase of the solvent’s refractive index upon
addition of GuHCl.
1 alpha 2 and 38 kDa-ribosome-associated protein
were able to interact with CP12 but to a lesser extent
than PRK, GAPDH and the aldolase, enzymes from
the Calvin cycle [9]. CP12 thus belongs to the IDP
family and it seems to be a jack of all trades and a
master of the Calvin cycle [15]. This protein is found
in most photosynthetic organisms [3,42] and was even
shown to be produced by cyanophages upon infection
of their hosts, thereby inhibiting their Calvin cycle and
re-routing the NADPH towards phage nucleotide biosynthesis [43]. A recent analysis in cyanobacteria
3211
CP12 structural modulation
S. B. Moparthi et al.
Auto correlation g (t)
A 1.008
CP12C31S - 0M GuHCl
1.006
CP12C31S - 5M GuHCl
CP12C75S - 0M GuHCl
1.004
CP12C75S - 5M GuHCl
1.002
1.000
0.1
1
10
Time (ms)
Hydrodynamic radii (nm)
B
6
CP12C75S
5
4
CP12C31S
3
2
Control
1
0
1
2
3
4
5
GuHCL concentration (M)
Fig. 6. Conformational changes of CP12 mutant proteins are
highlighted by unfolding experiments under denaturation
conditions. (A) Raw fluorescence correlation functions of CP12
mutant proteins in the presence and absence of GuHCl; the data
were fitted to a single-component diffusion model. (B)
Hydrodynamic radii of CP12 mutant proteins as function of the
concentration of GuHCl. A control experiment is displayed for
Atto647N free dye to indicate the absence of experimental
artifacts.
showed that about eight types of CP12 are present and
among them are three CP12-CBS containing a cystathionine-b-synthase (CBS) domain that is fused to
CP12. The CBS domains function as regulatory modules for a wide range of cellular activities, and some
bind adenine nucleotides [42]. The role of CP12 like
that of many other IDPs in the cell is thus of paramount importance [44].
Although the interaction between this protein, GAPDH and PRK has been studied, the structure and
dynamics of CP12 are still unknown. Crystallographic
data of GAPDH/CP12 from Synechococcus elongatus
and Arabidopsis thaliana showed that the C-terminal
part of CP12 can fold upon binding to GAPDH but
most of the CP12 remains highly flexible and almost
50 residues of about 80 were not visible in the density
map of either structure [10,45]. In C. reinhardtii it was
recently shown by site-directed spin labeling combined
with electron paramagnetic resonance spectroscopy
3212
that the GAPDH–CP12 complex is a fuzzy complex
[26]. Only a modeled structure of the algal CP12 is
therefore available [46]. Data on the interaction
between PRK and CP12 are scarce as this interaction
is weaker (lM range) than that with GAPDH and
CP12 (nM range) in green alga, and again no structural
data are available [2]. Hence not much is known on
the conformational change of the CP12 protein either
upon folding or upon interaction with GAPDH and
PRK proteins.
Our study focuses for the first time on structural
transition parameters of CP12–GAPDH and CP12–
PRK complexes. Our results indicate that the CP12wt
binds to GAPDH and/or PRK independently and also
to both enzymes to form a ternary complex. To understand the molecular background of the function of
CP12wt it is crucial, due to the high flexibility and the
lack of rigid structure of CP12, to investigate its structural properties. Since there are no structural data for
the algal complex, our present results attempt to elucidate the algal CP12 structure in terms of hydrodynamic radii to decipher its mode of binding to two key
regulated enzymes, namely GAPDH and PRK of the
Calvin cycle. These observations are a first step
towards an understanding of protein–protein interactions in the GAPDH–CP12–PRK complex and the
nature of the physicochemical forces involved during
ternary complex formation.
Our results showed that the disruption of the N-terminal disulfide bridge on CP12 affects the formation
of the CP12C31S–PRK complex, while it has no effect
on the CP12C31S–GAPDH complex. On the other
hand, we observed the opposite effect with the mutant
CP12C75S affected at the C-terminal disulfide bridge
that forms a complex with PRK but not with
GAPDH. Further addition of PRK or GAPDH to the
corresponding CP12C31S–GAPDH or CP12C75S–
PRK complexes showed no signs of the formation of
GAPDH–CP12(C31S/C75S)–PRK
supramolecular
complex suggesting that both C-terminal and N-terminal disulfide bridges are crucial for formation of the
ternary structure complex. In addition, the binding of
both GAPDH and PRK proteins to CP12 was suppressed by DTT that results in the disruption of the
disulfide bridges of CP12.
This study also showed that GAPDH and PRK bind
at specific sites on CP12. Based on the similar fluorescence brightness found for CP12 mutant proteins alone
and for the sub-complexes, only one monomer of
CP12C31S or CP12C75S bound to GAPDH and PRK
respectively. The mutant CP12C31S behaves as CP12
locked in a reduced state and therefore this result is in
agreement with previous results where only one moleFEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
S. B. Moparthi et al.
cule of reduced CP12 was bound to GAPDH while two
molecules of oxidized CP12 were bound to tetrameric
GAPDH [47,48]. The present study also indicates that
the ratio 1 : 4 of CP12 to PRK is sufficient to form the
CP12C75S–PRK complex; with a KD of 1.6 lM [2]
determined by surface plasmon resonance (SPR) and
the experimental conditions used in this report, one
would expect about 20% of complex formation between
CP12 and PRK. SPR and fluorescence spectroscopy are
complementary techniques although in SPR one partner
is immobilized on a chip while in FCS the interaction
between the two partners occurs in solution, explaining
the apparent discrepancy in the results obtained.
A set of equations correlating protein density in several conformational states (native, unfolded, pre-molten
globular and molten globular) in a wide range of molecular masses has been reported [49]. Analysis of similar
dependences for various IDPs was added later [18,50].
The measured hydrodynamic radii for both CP12
mutant proteins in their native state were around 32 A,
a value that fits more with an IDP hydrodynamic radius
compared with the native state hydrodynamic radius.
To cite but a few examples, the hydrodynamic radius
for a globular protein like cytochrome c is about 17.8 A
and for an IDP like TyrRS(D1) is 21 A, while these
proteins have almost the same number of amino acids
(104 and 107 respectively) [51]. Moreover, a clear difference was found between the extended conformation
(maximal chain expansion) under denaturing conditions
(Fig. 4A) and under native conditions. CP12 is highly
flexible in its native state and may become somewhat
ordered upon interaction with either GAPDH or PRK
by forming the GAPDH–CP12 or the PRK–CP12 binary complexes as observed by the diffusion time
changes upon formation of these sub-complexes.
The present report also focuses on the unfolding
nature of the CP12 mutation sites specifically both at
the C-terminus and N-terminus to assess the degree of
re-modulation and further to evaluate the specificity
towards the GAPDH and PRK interactions. It is likely
that CP12 like other IDPs shows gradual hydrophobic
collapse during unfolding.
Experimental procedures
Recombinant protein expression and purification
C. reinhardtii CP12, GAPDH and PRK were obtained as
described earlier [7]. The concentrations of all proteins were
calculated using Bradford assays [52]. The purity of recombinant proteins was checked by SDS/PAGE [53] followed by
Coomassie Blue R-250 staining and mass spectrometry
(Fig. 1C). For all experiments, His-tagged CP12 proteins
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
CP12 structural modulation
were used as it was shown that wild-type His-tagged CP12
behaves like the native protein, indicating that the His tag
does not interfere in our experiments [48]. Numbering of residues, however, was based on the protein without the tag,
with SGQPA being the first residues (http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Chlre4&id=148487).
Protein labeling
Lysine residues in the CP12wt protein were labeled with the
amine-reactive N-hydroxysuccinimide ester derivative of the
Atto590 fluorophore, and in both single cysteine-modified
CP12 mutant proteins were labeled with the thiol-reactive
maleimide derivative of the fluorophore Atto647N by
following the protocol provided by the manufacturer (AttoTec). In all cases, excess fluorophore was added to the aqueous protein solution containing 50 mM Tris/HCl at pH 8.0
and left for 8 h at 4 °C. The labeled protein was subsequently purified using size exclusion chromatography on a
SephadexTM G-25 Medium (GE Healthcare, Little Chalfont,
Buckinghamshire, UK) column with 50 mM Tris/HCl (pH
8.0) elution buffer. In this report, CD experiments were not
carried out as it was not possible to obtain enough labeled
protein (10 lM). However, previous reports showed that cysteine labeling of the proteins mutated either on the N-terminal disulfide bridge [26] or on the C-terminal disulfide bridge
[54] did not alter the global behavior of the CP12 proteins.
CD spectra of labeled and unlabeled proteins were similar.
Sample purity and specificity was confirmed by matrixassisted laser desorption/ionization mass spectrometry.
In vitro reconstitution of the GAPDH–CP12–PRK
complex
All in vitro reconstitution assays to test formation of the
sub-complexes CP12wt and CP12 mutants (C31S or C75S)
with GAPDH or PRK, or the ternary complex GAPDH–
CP12(wt or C31S or C75S)–PRK were performed as previously described [7]. In the case of GAPDH–CP12 or CP12–
PRK interaction experiments, 100 nM of labeled CP12wt or
CP12C31S or CP12C75S was mixed separately or in combination with 500 nM of GAPDH and 500 nM of PRK in
50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD and 5 mM
cysteine at pH 8.0 for 12 h at 22 °C. In the case of
CP12wt, the mixture was diluted to 10 nM of CP12wt and
50 nM of GAPDH or PRK respectively. In corresponding
samples, 1 mM DTT was used as a reducing agent and samples were incubated for 2 h at 22 °C. In the case of the
CP12 self-association experiments, 40 nM of labeled
CP12C31S mutant was mixed with ratios 1 : 12.5 nM,
1 : 125 nM and 1 : 1250 nM of unlabeled CP12C31S mutant
in 50 mM Tris/HCl, 4 mM EDTA at pH 8.0 for 12 h at
22 °C. To test the effect of PRK concentration on CP12–
PRK complex formation, 100 nM labeled CP12C75S was
mixed with concentration ratios from 1 : 0.04 nM to
3213
CP12 structural modulation
S. B. Moparthi et al.
1 : 20 nM of unlabeled PRK for 12 h at 22 °C in 50 mM
Tris/HCl, 4 mM EDTA, 0.1 mM NAD at pH 8.0. All the
mixtures were tested for complex formation using in vitro
reconstitution assays by electrophoresis under non-denaturing conditions (native PAGE) followed by Coomassie Blue
R-250 staining (data not shown).
Fluorescence correlation spectroscopy
FCS experiments were carried out with a custom-built confocal fluorescence microscope with a Zeiss C-Apochromat
40 9 1.2NA water-immersion objective [55]. The excitation
source was a CW He-Ne laser operating at 633 nm. For
FCS measurements on CP12 with either GAPDH or PRK
proteins the laser beam was set to fill the microscope objective back-aperture so as to use the maximum numerical
aperture available for the microscope objective. A 30 lm
confocal pinhole conjugated to the sample plane defined the
confocal volume whose transversal waist wxy was calibrated
to 285 nm using the known diffusion coefficient of Alexa
647 in pure water (3.1 9 106 cm2s1 at 22 °C). For the
experiment on CP12 unfolding, the increase in the refractive
index of the medium upon addition of GuHCl (Fig. 5) was
imposed to limit the negative effects of spherical aberrations.
This was achieved by under filling the microscope objective
back-aperture (4.5 mm instead of 8.9 mm) and using a
50 lm confocal pinhole. With these conditions, the transversal waist wxy was calibrated to 410 nm in the absence of GuHCl. A moderate variation of 20% on the diffusion time
was found with the calibration procedure using Alexa Fluor
647 upon increasing concentrations of GuHCl. This phenomenon was due to the combination of both refractive
effects and increased solvent viscosity g (Fig. 5). The effect
was systematically taken into account to calibrate the quotient wxy2/g in the presence of GuHCl so as to deduce the
hydrodynamic radius of the protein samples.
All FCS experiments were carried out at 22 °C using
50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD and 5 mM
cysteine as a reconstitution buffer at pH 8.0. We also
added 0.1% Tween-20 (Sigma) to the samples in order to
diminish surface interactions with the glass coverslip. The
fluorescence intensity temporal fluctuations were analyzed
with a hardware correlator (Flex02-12D/C correlator.com,
Bridgewater, NJ, USA, with 12.5 ns minimum channel
width). All the experimental data were fitted by considering
a single species and free Brownian 3D diffusion in the case
of a Gaussian molecular detection efficiency:
g2 ðsÞ ¼ 1 þ
2 1
B
s
1
1 þ nT exp N
F
sT
1 þ ssd
1
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 þ s2 ssd
ð1Þ
where N is the average number of molecules in the focal
volume, F is the total fluorescence signal, B is the back-
3214
ground noise, nT is the amplitude of the dark state population, sT is the dark state blinking time, sd is the mean
diffusion time and s is the ratio of transversal to axial
dimensions of the analysis volume. The background noise
B originated mainly from the back-reflected laser light and
from the detector dark current and remained below 1 kHz
for the experiments reported here. Numerical fit of the FCS
data following Eqn (1) provided the average number of
molecules, N, which was used to calculate the fluorescence
count rate per molecule (CRM) as
CRM ¼
FB
N
ð2Þ
All the data were fitted by considering that the regular
distribution of the weighted residuals is around zero. The
FCS measurement of the mean diffusion time sd and the
separate calibration of the transversal waist wxy of the confocal detection volume allowed the molecular diffusion
coefficient D to be calculated according to the relation
sd ¼ w2xy =4D
ð3Þ
The hydrodynamic radius RH can then be deduced from
the measured diffusion coefficient D using the Stokes–Einstein equation
D¼
kB T
6 p g RH
ð4Þ
where kB is Boltzmann’s constant, T is the absolute temperature and g is the viscosity of the medium. We calibrated
the parameter w2xy /g before each measurement on CP12 by
recording the FCS trace for Alexa Fluor 647 dyes which
have a known hydrodynamic radius of 0.7 nm in pure
water. With that calibration, the measured hydrodynamic
radii of both Alexa Fluor 647 and Atto647N do not vary
upon addition of GuHCl, confirming our experimental
method.
Conclusion
We report detailed information about the hydrodynamic radii of wild-type CP12 and its site-specific
mutants at their disulfide bridges at the N-terminus
(CP12C31S) and the C-terminus (CP12C75S). We
quantify a hydrodynamic radius of 3.4 0.2 nm for
the CP12 protein with an increase up to 5.2 0.3 nm
upon complex formation with GAPDH and PRK. Our
results on CP12C31S and CP12C75S clearly point out
the crucial influence of the mutations affecting the
disulfide bridges for complex formation between CP12,
GAPDH and PRK. Using denaturation conditions, we
monitor the change in hydrodynamic radii of the
CP12 mutant proteins upon unfolding. The different
behavior between CP12 mutant proteins indicates
FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
S. B. Moparthi et al.
different structural conformations that favor the binding of either the GAPDH tetramer or the PRK dimer.
Acknowledgements
This work was funded by a contract from the European Research Council under the European Union’s
Seventh Framework Programme (FP7/2007-2013)/
ERC Grant agreement no. 278242. GP was in receipt
of a Fellowship from the Ecole Normale Superieure,
Cachan. This work (BG) was in part supported by the
Agence Nationale de la Recherche ANR SPINFOLD
no. 09-BLAN-0100, La Region PACA, APO projet
063 506, the Centre National de la Recherche Scientifique (CNRS) and Aix-Marseille Universite.
Author contributions
Design of the experiments: SBM, HR, BG and JW.
Involved in protein purification and labeling: SBM
and GPT. Involved in FCS experiments: SBM and
JW. Helped in protein quantification: PM. Analyzed
the data: SBM, GPT, BG and JW. Wrote the paper:
SBM, BG and JW. All authors read and approved the
final manuscript.
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