©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic Analysis of Interactions between GroEL and Reduced -Lactalbumin
EFFECT OF GroES AND NUCLEOTIDES (*)

(Received for publication, May 1, 1995; and in revised form, June 22, 1995)

Noriyuki Murai Hideki Taguchi Masasuke Yoshida (§)

From the Tokyo Institute of Technology, Research Laboratory of Resources Utilization, R-1, 4259 Nagatsuta, Yokohama 226, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The real-time analysis of the association and dissociation of chaperonin with respect to its substrate protein was carried out using the BIAcore


INTRODUCTION

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 (^1)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. alpha-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 alpha-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


EXPERIMENTAL PROCEDURES

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 alpha-Lactalbumin on Sensor Chips

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^2)(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(2), 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 .

where k 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 .

where RU(1) is a resonance unit at the time of changing the solution (time = t(1)), RU is that at time t, and t is t - t(1). Although the infinity point for the dissociation reaction should have been subtracted from RU(1) and RU(t), 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.


RESULTS

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 GroELbulletrLA interaction except that the presence of KCl was required when ATP was injected to induce dissociation (data not shown).


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.



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. (^2)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 10^5M 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.


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 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.


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.





Effect of ADP on the GroELbulletrLA interaction

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 k 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 GroELbulletrLA 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.


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.





Effect of ADP on GroEL/ESbulletrLA Interaction

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/ESbulletrLA 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/ESbulletrLA 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 10^3M 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) 10M. 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/ESbulletrLA 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 GroELbulletrLA was examined (Fig. 5A). The dissociation was initiated by changing the GroEL-containing buffer to the free buffer, (^3)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 GroELbulletrLA complex and the GroEL/ESbulletrLA complex probably by competing with rLA for the same sites on GroEL.


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^2). 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.




DISCUSSION

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 10^5M s) but not as fast as it binds to nonnative barnase (k = 1.3 10^8M s)(22) . Dissociation is very slow (k = 2 10 s), consistent with the fact that one can isolate the GroELbulletrLA complex with gel filtration chromatography (20, 21) . Both fast binding and slow release of GroEL contribute to the very small K value (1 10M). 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 10M) 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 (10-10M) of the interaction between GroEL and a peptide (beta-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 10^3 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.


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 GroELbulletrLA (or GroEL/ESbulletrLA) 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/ESbulletrLA complex was destabilized by an excess amount of GroES (Fig. 5). GroELbulletrLA was also destabilized. In the presence of excess GroES, GroELbulletrLA may be converted into GroEL/ESbulletrLA, which is then destabilized by another GroES. The mechanism of the GroES effect on the stability of GroEL/ESbulletrLA 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 GroESbulletGroELbulletGroES 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 GroESbulletGroELbulletGroES complex, which can no longer rebind to immobilized rLA. This GroES-dependent destabilization did not require adenine nucleotide. Without adenine nucleotide, the symmetric GroESbulletGroELbulletGroES 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/ESbulletrLA complex.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 81-45-924-5277.

(^1)
The abbreviations used are: GroEL/ES, an assembled form of GroEL and GroES; LA, alpha-lactalbumin; rLA, Ca-depleted alpha-lactalbumin whose four disulfide bonds are reduced; apo-LA, Ca-depleted alpha-lactalbumin; BSA, bovine serum albumin; RU, resonance units; AMP-PNP, adenosine 5`-(beta,-imino)triphosphate.

(^2)
This involves apparent first-order kinetics and does not always indicate a single reaction pathway in which GroEL could interact with rLA. Rather, on a microscopic view, there can be multiple pathways with similar rate constants. The same precaution should be observed in interpreting other rate constants in this paper.

(^3)
In this experiment, due to the technical limitation of the buffer change system of the BIAcore system, the running buffer used in the association phase could not be directly changed to the GroES-containing buffer but was changed first to the free buffer.


ACKNOWLEDGEMENTS

We thank Drs. I. Yahara and Y. Miyata for the generous offer to use their BIAcore

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