(Received for publication, May 1, 1995; and in revised form, June 22, 1995)
From the
The real-time analysis of the association and dissociation of chaperonin with respect to its substrate protein was carried out using the BIAcore
Two chaperonin proteins of Escherichia coli, GroEL and
GroES, collaboratively play an essential role in assisting the folding
of proteins in the cell(1, 2) . As determined by x-ray
crystallography, GroEL appears to be a porous cylinder of 14 identical
57-kDa subunits and is made of 7-fold rotationally symmetrical rings
stacked back-to-back with a dyad symmetry(3) . Co-chaperonin,
GroES, is a heptameric ring of identical 10-kDa subunits. These
chaperonins form two types of heterooligomers in vitro; in the
presence of ADP, a heptamer ring of GroES can bind to one or both
termini of a single GroEL tetradecamer cylinder to form the GroEL/ES (
where k
where RU
Figure 1:
Binding of GroEL to immobilized LA. Trace A, rLA was immobilized directly on the research grade
sensor chip surface at the level of 3500 RU. GroEL (50 nM, as
14-mer) was injected successively for 11 min over the immobilized rLA,
and the binding was monitored by the increase in the RU level. The
dissociation phase was initiated by switching the flow solution to the
free buffer. The sensor chip surface was regenerated with the buffer
containing 1 mM ATP. Trace B, native LA was
immobilized directly on the sensor chip surface at the level of 3000
RU, and GroEL (75 nM) was injected. The time points of the
injection are indicated by arrows and letters.
Figure 2:
Kinetics of GroEL binding to immobilized
rLA. A, overlay plot of the binding of GroEL to immobilized
rLA (3500 RU on a research grade sensor chip) at various concentrations
of GroEL. GroEL was injected over immobilized rLA for 11 min at a
concentration of 50, 38, 25, 13, or 6 nM (from the cycle at
the top, as 14-mer). The dissociation phase was started by switching
the flow solution to the free buffer. The sensor chip surface was
regenerated with the buffer containing 1 mM ATP. The time
points of the injection are indicated by arrows and letters. B, plot of slope versus the
concentration of GroEL. The slope was determined from the plot of the
first derivative (dRU/dt) versus RU(t) of the traces in A for the periods when
binding was not limited by mass transfer(29) . C, plot
of the dissociation phase from t = 835-1015 s.
Dissociation is expressed as the natural log (ln) of the drop in RUversus time. The plots for GroEL bound at 50, 38, 25, 13,
and 6 nM are shown (from the upperline). Inset, plot of k
Figure 3:
Effect of adenine nucleotides on
dissociation of (A) GroEL and (B) GroEL/ES from
immobilized rLA. A, sensorgram of the dissociation phase of
GroEL from immobilized rLA (2000 RU on a research grade sensor chip).
GroEL (75 nM as 14-mer) was injected over immobilized rLA
successively for 7 min in which time the increase in RU almost leveled
off. The dissociation phase was initiated by switching the flow
solution to the buffer containing none, 1 mM AMP, 1 mM AMP-PNP, 1 mM ADP, or 1 mM ATP at the time
indicated by an arrow. The magnitude of the net RU increase
from the base line at the time of initiation of the dissociation phase
was taken as 100% for each sensorgram. B, sensorgram of the
dissociation of GroEL/ES from immobilized rLA (2000 RU on a research
grade sensor chip). GroEL (75 nM as 14-mer) and GroES (225
nM as 7-mer) were preincubated with 10 µM ADP for
1 h at 25 °C to form the GroEL/ES complex and used for the
experiments. Other conditions were the same as in A.
Figure 4:
Effect of ADP concentrations on binding of
GroEL and GroEL/ES to the immobilized rLA. A, binding of GroEL
(75 nM, as 14-mer) in the presence of ADP. GroEL was
separately incubated in the buffer containing 0, 10, 100, and 500
µM and 1 mM ADP for 1 h at 25 °C. After the
incubation, these samples were injected over immobilized rLA (3500 RU
on a research grade sensor chip). The dissociation phase was initiated
by switching to the free buffer. The surface of immobilized rLA was
regenerated with the free buffer containing 1 mM ATP. B, binding of GroEL/ES in the presence of ADP. GroEL (75
nM, as 14-mer) and GroES (225 nM, as a 7-mer) were
mixed and incubated in the buffer containing 0, 10, 20, 30, 50, 70,
100, and 250 µM and 1 mM ADP for 1 h at 25
°C. After the incubation, these samples were injected over
immobilized rLA as indicated. Other conditions were the same as those
of A. Inset, the relative amount of GroEL (RU) bound to
immobilized rLA was plotted against the concentration of ADP. RU values
immediately before initiation of the dissociation phase were taken as
representative values of the binding. The same sensor chip surface of
immobilized rLA was used for the experiments in A and B.
Figure 5:
Effect of excess GroES on dissociation of
GroEL and GroEL/ES from immobilized rLA. A, after GroEL (75
nM) was injected over immobilized rLA (2000 RU on a research
grade sensor chip) for 7 min, the flow solution was changed to the free
buffer and then to the buffer containing the indicated concentration of
GroES or BSA. The surface of immobilized rLA was regenerated with 1
mM ATP. B, GroEL (75 nM) and GroES (225
nM) were mixed, incubated with 10 µM ADP for 1 h
at 25 °C, and injected over immobilized rLA. Other conditions were
the same as those of A. C, GroEL (157 nM) was
injected over immobilized rLA (800 RU = 0.8 ng/mm
Figure 6:
Scheme for the effect of ADP on the
kinetics of GroEL and GroEL/ES in association and dissociation with
respect to substrate protein. The rates of association are expressed as
time required for the association of half the population of GroEL (or
GroEL/ES) with rLA when 1 µM GroEL (or GroEL/ES) and 1
µM rLA are mixed. The rates of dissociation are expressed
as time required for the dissociation of half of the population of
GroEL
)complex(4, 5, 6) , and in the
presence of ATP, two heptamer rings of GroES can bind to both termini (7, 8, 9, 10) . GroEL binds
nonnative proteins that are structurally unrelated in their native
state (11) and prevents
aggregation(12, 13, 14) . It has been
suggested that, in the presence of GroES, GroEL functions under the
cycles of binding and release of nonnative proteins coupled with ATP
hydrolysis that is
K
-dependent(10, 15) . Substrate
proteins released from chaperonin are kinetically partitioned to
folding to the native state, to aggregation, or to rebinding to another
chaperonin molecule(10, 15, 16) . To date,
many proteins are known to interact with the GroEL/ES.
-Lactalbumin (LA) is one of these proteins whose several forms
with different folding states have been well characterized. This
protein is a 123-residue Ca
-binding
protein(17) , and Ca
-depleted LA (apo-LA)
assumes a state of molten globule at neutral
pH(18, 19) . When the four disulfide bonds in
-lactalbumin are reduced (rLA), the protein structure becomes more
expanded than apo-LA, and GroEL can bind rLA but not
apo-LA(20, 21) . Including the above studies, several
studies analyzed the affinity and kinetics of the interaction between
GroEL/ES and substrate proteins(22, 23) . However,
they are all indirect measurements, and there are only a few studies
analyzing the association and dissociation process directly. Recently,
an instrument called the BIAcore
Materials
GroEL and GroES were purified from
overexpressing E. coli cells as described
previously(24) . The concentrations of GroEL and GroES are
expressed as 14-mer and 7-mer, respectively, throughout this paper.
Three forms of bovine LA were obtained from Sigma (type I, native form;
type III, Ca-depleted form; reduced S-carboxymethylated form). rLA was prepared by incubating LA
(type III) with 2 mM dithiothreitol(20) . Protein
concentrations were assayed by the method of Bradford with bovine serum
albumin (BSA) as the standard(25) .
BIAcore Analysis
The principle of the BIAcore
system (Pharmacia Biotech Inc.) is based on a quantum mechanical
phenomenon that is called surface plasmon
resonance(26, 27) . This phenomenon is as follows. The
light enters a highly refractive index glass at one angle of incidence
such that the light reflects totally on the boundary surface between
the glass and the outside of the material layer; then, if a material
layer is touching the reflecting surface, the light entering at one
angle of incidence is absorbed and therefore the intensity of the
catoptric light is reduced. The angle such that the intensity of
reflection is reduced is dependent on the refractive index of the
medium touching the outside of the metal layer. When the light reflects
totally on the boundary surface between the glass and the material
layer, a small wave called an evanescent wave, which is conveyed only
on the surface, is generated. On the other hand, a surface wave called
surface plasmon resonance occurs on the surface of the metal layer.
When these two surface wave numbers coincide, resonance occurs, and the
intensity of the catoptric light is reduced because a portion of the
optical energy is used to excite the surface plasmon resonance. Surface
plasmon resonance is affected only by the medium near the metal layer.
Thus the incidence angle that reduces the intensity of the catoptric
light is changed if a concentration change in the medium occurs near
the metal layer. This change reflects the amount of material changes in
the solution touching the sensor surface. Actually for the sensor chip,
a dextran matrix is mounted on the top of a thin gold layer, which is
placed on the supporting glass layer. The protein can be immobilized on
this matrix by covalent coupling, and the sensor chip is placed onto
the optical unit thus creating a flow cell over this matrix.Immobilization of
Immobilization of the native LA, rLA, and reduced S-carboxymethylated-LA was attempted directly onto the dextran
matrix of the sensor chip (CM5) surface by covalent coupling of primary
amine groups on LA to carboxyl groups on the dextran matrix of the
sensor chip. The carboxylate dextran matrix of the sensor chip was
activated with 35 µl of a mixture of N-ethyl-N`-(3-dimethylamino)carbodiimide
hydrochloride and N-hydroxysuccinimide. Two forms of LA were
injected at a concentration of 1.0 mg/ml (native LA) or 1.1 mg/ml (rLA)
in 10 mM sodium acetate buffer (pH 4.0) and 2 mM dithiothreitol (in the case of rLA) at a flow of 5 µl/min at
25 °C. Usually, 800-3500 resonance units (RUs) were immobilized.
The resonance unit (RU) is an arbitrary unit used in the BIAcore
system, and there is a linear relationship between the mass of the
protein bound to the dextran matrix and the RU observed (1000 RU
= 1 ng/mm-Lactalbumin on Sensor
Chips
)(28, 29) . In the case of
native LA, the protein was immobilized onto the sensor chip surface
immediately after it was dissolved in the acetate buffer to prevent
denaturation, and 3000 RUs of native LA were immobilized. Reduced S-carboxymethylated LA (3 mg/ml) precipitated at pH 4.0 in the
acetate buffer used for immobilization. At pH 4.8, it remained soluble,
but no immobilization was achieved at this pH. The remaining
carboxylate groups on the sensor chip were blocked by a subsequent
injection of 35 µl of 1 M ethanolamine-HCl buffer (pH
8.5).
Binding Kinetics
The buffer used for the flow
(free buffer) was 10 mM HEPES buffer (pH 7.4) containing 150
mM KCl, 20 mM MgCl, and 2 mM dithiothreitol. All of the nucleotides and proteins were dissolved
in the free buffer and used for the experiments. In the case of native
LA, dithiothreitol was omitted. Samples were injected at 25 °C with
a flow rate of 5 µl/min onto the sensor chip surface on which LA
had been immobilized. The traces of the association and dissociation
process were analyzed with BIAlogue30) . Briefly, a series of
different concentrations of rLA are injected, and the change in the RU
is converted automatically to a plot of RU versus time, called
a sensorgram, by the system software. The slopes of dRU/dt versus RU plots are calculated next,
and the slopes at each concentration are replotted against the
concentration of injected protein. The rate constant can be calculated
from .
and k
are
the association and dissociation rate constants, respectively, and C is the concentration of injected protein. The dissociation
rate constant is also calculated from the dissociation phase after the
buffer containing the protein is changed to the protein-free buffer.
The dissociation obeys first-order kinetics described by .
is a resonance unit at
the time of changing the solution (time = t
), RU
is that at
time t
, and t is t
- t
.
Although the infinity point for the dissociation reaction should have
been subtracted from RU
and RU
, we used the original level of the sensorgram
with the starting buffer as the level of the infinity point. The
dissociation constant (K
) was calculated
from the equation K
= k
/k
.
Binding of GroEL to Immobilized LA
When GroEL
was injected over the sensor chip with immobilized rLA, the RU level
increased up to about 4000 RU (Fig. 1, trace A). This
change in RU was not observed when the same concentrations of BSA and
ribonuclease were injected (data not shown). When the running solution
containing GroEL was switched to the free buffer after the RU change
was nearly saturated, GroEL started to dissociate from the immobilized
rLA only very slowly (spontaneous dissociation). Further injection of
the buffer containing 1 mM ATP induced rapid dissociation of
GroEL, ensuring that the association was specific. When GroEL was
injected over the sensor chip with immobilized native LA (Fig. 1, trace B), only a very slight increase in RU
called a ``bulk effect'' (31, 32) was
observed (Fig. 1, trace B). This effect is caused by
the presence of compounds different from those of the previous running
solution. This increase returned to the original level on changing the
solution to the free buffer. Thus, as expected from the results of
other groups(20, 21) , GroEL did not interact with the
native form of LA. Interaction of GroEL with reduced S-carboxymethylated LA was not tested because immobilization
onto the sensor chip was unsuccessful. Therefore, all of the following
experiments were carried out for rLA as an immobilized substrate
protein. All of the solutions used in the above experiments contained
0.15 M KCl. However, substitution of KCl with NaCl did not
change the essential features of the GroELrLA interaction except
that the presence of KCl was required when ATP was injected to induce
dissociation (data not shown).
Kinetic Analysis of Interaction between GroEL and
rLA
To estimate the rate constants, the running buffers
containing GroEL at various concentrations of 6-50 nM were injected, and this buffer was changed to the free buffer
after RU reached the saturation level (Fig. 2A). The dRU/dt versus RU plots in each of the sensorgrams in Fig. 2A were linear (data not shown), indicating that
association occurs as an apparent first-order reaction. ()The slopes of the dRU/dt versus RU plots
were then replotted against GroEL concentrations (Fig. 2B), and the association rate constant (k
) and dissociation rate constant (k
) were estimated according to .
The slope of the line in Fig. 2B gave a fairly fast k
value, 1.96
10
M
s
, and the y intercept showed a slow k
value, 8.51
10
s
. The k
value was also independently obtained from the
analysis of the dissociation phase of Fig. 2A according
to (Fig. 2C). The k
values calculated from the sensorgrams at lower GroEL
concentrations were more rapid than those at high GroEL concentrations,
probably due to the occurrence of rebinding of newly dissociated GroEL
to other vacant sites on rLA immobilized on the sensor
chip(33) . The value became nearly constant when the GroEL
concentration increased (Fig. 2C, inset), and
we adopted the value 2.08
10
s
that was calculated at the highest GroEL concentration tested as
the k
value. This value is equivalent to a decay
half-time of about 1 h. The k
value obtained
from the dissociation phase is 4 times slower than that obtained from
the association phase. In general, the k
value
obtained from the association phase is not very reliable when it is
low(30) ; therefore, we selected the k
value obtained from the dissociation phase as the true value.
From these rate constants, the dissociation constant (K
) is calculated to be 1.06 nM.
value versus the concentration of GroEL.
Dissociation by Adenine Nucleotides
The very slow
spontaneous dissociation of GroEL from immobilized rLA described above
was accelerated by ATP, ADP, and AMP-PNP (Fig. 3A, Table 1). ATP was the most effective, more than 3 orders of
magnitude acceleration from spontaneous dissociation, and ADP and
AMP-PNP were also able to stimulate dissociation. AMP had essentially
no significant effect on the dissociation. The same experiments were
done for the GroEL/ES, and similar results were observed (Fig. 3B, Table 1). When the preformed GroEL/ES
in the presence of 10 µM ADP was allowed to bind
immobilized rLA and various adenine nucleotides were then injected, the
dissociation of the GroEL/ES from the immobilized rLA was accelerated
by ATP, ADP, and AMP-PNP, but not AMP.
Effect of ADP on the GroEL
When GroEL was mixed with 10 µM, 100
µM, 500 µM, or 1 mM ADP, and the
mixtures were injected onto the sensor chip with immobilized rLA, the
association phase of each sensorgram did not appear to change
significantly from that of GroEL alone (Fig. 4A).
Actually, the krLA
interaction
values of GroEL to rLA in the
presence of 10 µM and 1 mM ADP obtained from a
series of experiments with different GroEL concentrations (data not
shown) indicated that association was still very fast in the presence
of ADP (Table 2). In the absence of ADP, the dissociation of the
complexes, which had been formed in the presence of ADP, was not very
different from that of GroEL alone (Fig. 4A). The
stability of the GroEL
rLA complex was relatively indifferent to
the presence or absence of ADP at the time of the complex formation. To
calculate k
and k
values
in the presence of ADP, another series of experiments was carried out
in which all of the solutions used in the sensorgram contained the same
concentration of ADP (data not shown). As shown in Table 2, the
rate of dissociation in 1 mM ADP was 2 orders of magnitude
faster than the absence of ADP. This accelerated dissociation by ADP
was observed only at a high concentration of ADP. The k
in the presence of 10 µM ADP was
not significantly different from the k
in the
absence of ADP. The dissociation constant K
in 1 mM ADP was 2 orders weaker (larger) than in
the absence of ADP. To summarize, what is drastically changed in the
interaction between GroEL and rLA by the presence of a high
concentration of ADP is not the association rate but the dissociation
rate which is accelerated more than 100 times.
Effect of ADP on GroEL/ES
Similar experiments were done for GroEL/ES. As
reported(34) , we confirmed by gel filtration HPLC that
GroEL/ES was formed in the presence of ADP at 10 µM and 1
mM (data not shown). Therefore, GroEL and GroES were at first
incubated for 1 h at 25 °C in the presence of various
concentrations of ADP to allow formation of the GroEL/ES, and the
incubated solutions were then injected onto the sensor chip with
immobilized rLA. As shown in Fig. 4B, the
association-dissociation kinetics were similar to those in the absence
of ADP when the concentration of ADP was 10 and 20 µM.
However, as the concentrations of ADP were increased over 30
µM, the RU response of the sensorgram started to decrease
and disappeared completely at 250 µM ADP. The ADP
concentration that gave a half-maximum effect was about 40 µM (Fig. 4B, inset). Although the rate
constants in the presence of 1 mM ADP cannot be calculated
because only a very slight amount of GroEL/ESrLA
Interaction
rLA was formed (see
the sensorgram in Fig. 4B), one can roughly
estimate the largest possible k
value on the
assumption that k
in the presence of 1 mM ADP is the same when the GroEL/ES
rLA complex is formed at 10
µM and 1 mM ADP. If k
is
(5.5 ± 1.5)
10
s
,
then the extent of very poor association observed in the presence of 1
mM ADP can be used for the simulation to set an upper limit on k
. When the k
value is
smaller than 1.0
10
M
s
, a computer-simulated sensorgram (not shown)
becomes similar to that observed in the experiment in Fig. 4B.
The K
is then estimated to be larger than
(5.5 ± 1.5)
10
M. These
results imply that there are two kinds of ADP binding sites with
different affinities on the GroEL/ES; occupation of the first high
affinity sites by ADP is necessary for formation of the GroEL/ES, and
occupation of the second relatively low affinity sites by ADP results
in the loss of ability of the GroEL/ES to retain nonnative substrate
proteins. Occupation of the second sites by ADP occurred rapidly,
because a 2-min preincubation, the minimum period in our experiments,
instead of 1 h, gave the same results. When 0.15 M KCl in the
buffer was substituted with 0.15 M NaCl, a very similar effect
of ADP was observed, except that the ADP concentration that gave a
half-maximum effect on the GroEL/ES
rLA interaction was about 400
µM rather than 40 µM (data not shown).
GroES stimulates dissociation of GroEL and the GroEL/ES
from rLA
The effect of excess GroES on the stability of the
GroELrLA was examined (Fig. 5A). The dissociation
was initiated by changing the GroEL-containing buffer to the free
buffer, (
)and then the solution containing GroES was
injected for 5 min. As the concentration of GroES increased from 0.1 to
5 µM, the slopes of the dissociation became steeper. The
same concentration of BSA caused only a slight effect. The same
experiments were carried out for the GroEL/ES. In this case, ADP at 10
µM was contained in all of the solutions to form and
stabilize the GroEL/ES. The results were almost the same as those of
GroEL (Fig. 5B). This effect of GroES on the
dissociation of GroEL was also observed in the solutions containing
0.15 M NaCl instead of KCl (Fig. 5C). In the
NaCl solution, a nonspecific effect by BSA was completely suppressed,
but the effect of GroES was as obvious as in the experiments in KCl
buffer. These results suggest that even in the absence of a nucleotide,
GroES can destabilize both the GroEL
rLA complex and the
GroEL/ES
rLA complex probably by competing with rLA for the same
sites on GroEL.
).
The indicated concentration of GroES or BSA was injected after the free
buffer. Other conditions were the same as those in A except
that all of the buffers contained 0.15 M NaCl instead of 0.15 M KCl.
Chaperonin Binds to Substrate Protein Very
Tightly
In this study, the association and dissociation of
chaperonin with respect to a substrate protein were monitored in real
time using a novel biosensor, the BIAcore system. GroEL binds to rLA
fast (k =
2
10
M
s
) but not as
fast as it binds to nonnative barnase (k
=
1.3
10
M
s
)(22) . Dissociation is very slow (k
=
2
10
s
), consistent with the fact that one can
isolate the GroEL
rLA complex with gel filtration chromatography (20, 21) . Both fast binding and slow release of GroEL
contribute to the very small K
value
(
1
10
M). As represented by
this very small K
value, the interaction
between GroEL and rLA is very strong, comparable with the strongest
antigen-antibody interaction (35) . This value is close to the K
value (7
10
M) of the interaction between GroEL and nonnative
lactate dehydrogenase, which was indirectly estimated from the ability
to retard the refolding of the enzyme (36) but smaller than the K
value
(10
-10
M) of the
interaction between GroEL and a peptide (
-lactamase presequence),
which was measured with the BIAcore system, probably because of the
small size of the substrate peptide(33) . As expected,
dissociation is greatly stimulated by ATP, more than 10
times. Interestingly, ADP and AMP-PNP can accelerate the
dissociation, although less efficiently than ATP, and, therefore, ATP
hydrolysis is not absolutely required for the dissociation (Fig. 3). This is also the case for the GroEL/ES. These
observations are consistent with the results that ADP and other
nucleotides can resume folding when added to the GroEL/ES-arrested
nonnative proteins(37, 38) .
Effect of ADP on GroEL and GroEL/ES
The effects of
low and high concentrations of ADP described in Fig. 4and Table 2are reasonably explained based on the scheme illustrated
in Fig. 6. GroEL and GroEL/ES show an apparently similar
response to ADP. The addition of 10 µM ADP does not have a
significant effect on the kinetics, but 1 mM ADP induces a
100-fold increase in k values (Table 2).
As for GroEL, the results are well interpreted, assuming that it has
only low affinity ADP binding sites. ADP at a concentration of 10
µM does not have an effect on kinetics probably because
ADP cannot bind to GroEL. With 1 mM ADP, low affinity ADP
binding sites are occupied, and GroEL dissociates from immobilized rLA (Fig. 3A). The minimum ADP concentration causing
dissociation was not determined, but it may be around or above 0.5
mM, because Todd et al.(10) reported that
0.5 mM ADP does not significantly stimulate the release of
Rubisco from GroEL(10) . Contrary to GroEL, GroEL/ES has two
kinds of ADP binding sites. The high affinity ADP binding sites (ADP* in Fig. 6) are the same sites that are
responsible for stabilizing GroEL/ES(34) . Interactions between
GroEL/ES and rLA are affected very little, if any, by occupation of the
high affinity sites by ADP (Table 2). When the low affinity ADP
binding sites of GroEL/ES, which are saturated at 70 µM ADP (Fig. 4B, inset), are occupied,
dissociation of GroEL/ES from immobilized rLA is greatly accelerated.
Because the binding of ADP is thought to be very fast and cannot be the
rate-limiting step in the dissociation kinetics of the substrate
protein, when B` is exposed to an ADP-free solution,
dissociation of GroEL occurs only slowly through pathway B`
A`
A (Fig. 4A shows the dissociation
phase of the sensorgram at 1 mM ADP). Similarly, if D` is
exposed to 10 µM ADP solution, GroEL/ES probably
dissociates through pathway D`
C`
C, but this has not
been confirmed by experiment due to lack of the D` population to
initiate the reaction. The rate of association is affected by ADP in a
different manner for GroEL and GroEL/ES. Association rate constants of
GroEL in the presence of 1 mM ADP are 30% of that in the
absence of ADP in the case of GroEL and less than 1% in the case of
GroEL/ES. For GroEL/ES, upon occupation of the low affinity ADP binding
sites, not only the dissociation phase but also the association phase
is changed in an unfavorable direction for binding of rLA. Therefore,
the GroES moiety in GroEL/ES plays a role in regulation of the rate of
association in response to the state of the second nucleotide binding
sites. This postulation is consistent with the recent report by Todd et al.(10, 39) . They suggested that the
GroEL/ES has the ability to bind substrate protein when the seven
adenine nucleotide binding sites of one of two GroEL heptamer rings are
occupied by ADP, but that the ability is diminished (or lost) when
another seven adenine nucleotide binding sites of the other GroEL
heptamer ring are occupied by ADP. The GroEL/ES all of whose adenine
nucleotide binding sites are occupied by ADP (D in Fig. 6) is likely to be an inactive form.
rLA (or GroEL/ES
rLA) complexes. Calculations are based
on values listed in Table 1and Table 2. GroEL and GroES
are shown as the white and shadowedboxes,
respectively. A string with an irregular shape attached on the bottom
of GroEL represents the substrate protein (rLA). ADP*
indicates the ADP molecule bound at high affinity nucleotide binding
sites. Thick and thinarrows indicate fast
and slow reactions, respectively. Binding and release of ADP are
thought to be very fast except for the release of ADP*, which is very
slow in some instances (10) . When the ADP concentration is
higher than 100 µM, all of the GroEL/ES population exists
in the state of D, which is almost inactive with respect to the ability
to interact with the substrate protein.
Competition for the Same GroEL Site by rLA and
GroES
The GroEL/ESrLA complex was destabilized by an
excess amount of GroES (Fig. 5). GroEL
rLA was also
destabilized. In the presence of excess GroES, GroEL
rLA may be
converted into GroEL/ES
rLA, which is then destabilized by another
GroES. The mechanism of the GroES effect on the stability of
GroEL/ES
rLA is not known. If GroES competes with the substrate
polypeptide for the same or overlapping binding sites on GroEL as
implicated by structural and mutational analysis(40) , one can
speculate that these sites, where rLA binds, are deprived by GroES in
the presence of excess GroES. Probably, rLA does not occupy all of the
seven binding sites of GroEL/ES at a given moment, and GroES binds to a
vacant site at first, occupies the sites one by one, and finally all of
the binding sites are taken by GroES, releasing the free symmetric
GroES
GroEL
GroES complex. Another explanation is that very
frequent release-rebinding of GroEL/ES to immobilized rLA is occurring
on the surface of a sensor chip, and GroES binds to the free GroEL/ES
to form the GroES
GroEL
GroES complex, which can no longer
rebind to immobilized rLA. This GroES-dependent destabilization did not
require adenine nucleotide. Without adenine nucleotide, the symmetric
GroES
GroEL
GroES complex may rapidly decay to GroEL/ES or
even to GroEL, which can rebind to immobilized rLA. This might be the
reason why a large molar excess of GroES is required for apparent
destabilization of the GroEL/ES
rLA complex.