©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stability of the Asymmetric Escherichia coli Chaperonin Complex
GUANIDINE CHLORIDE CAUSES RAPID DISSOCIATION (*)

Matthew J. Todd (§) George H. Lorimer

From the E. I. DuPont de Nemours & Co., Central Research and Development Department, Experimental Station, Wilmington, Delaware 19880-0402

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The chaperonin proteins, GroEL(14) and GroES(7), inhibit protein aggregation and assist in protein folding in a potassium/ATP-dependent manner. In vitro, assays for chaperonin activity typically involve adding a denatured substrate protein to the chaperonins and measuring the appearance of correctly folded substrate protein. The influence of denaturant is generally ignored. Low concentrations of guanidinium chloride (<100 mM) had a profound effect on the activity/structure of the chaperonins. Guanidinium decreased the ATPase activity of GroEL and attenuated the inhibition of GroEL ATP hydrolysis by GroES. The stable, asymmetric chaperonin complex which forms in the presence of GroES and ADP (GroES(7) bullet ADP(7) bullet GroEL(7)-GroEL(7)) rapidly dissociated upon addition of 80 mM guanidinium chloride. Dissociation was enhanced at high ionic strength, but rapid dissociation was guanidinium-specific. Accelerated release of the GroES from the complex was also demonstrated. Unfolded proteins alone had no effect on complex stability. Residual guanidinium depressed the rate of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase (Rubisco) folding; an increased aggregation rate also decreased the yield of folded Rubisco. Chaperonin-assisted folding is therefore best studied using proteins denatured by means other than guanidinium chloride.


INTRODUCTION

Molecular chaperones are ubiquitous proteins which assist in the transport, folding, and assembly of other proteins (Lorimer, 1992; Gatenby and Viitanen, 1994). Two Escherichia coli chaperone proteins, GroEL and GroES, exist as double and single heptameric rings, respectively (Hendrix, 1979; Chandrasekhar et al., 1986). These proteins have been termed chaperonins (Ellis, 1992) and assist in protein folding, both in vivo and in vitro (Goloubinoff et al., 1989a, 1989b).

The GroEL protein stably binds one (Laminet et al., 1990) or two (Viitanen et al., 1991) unfolded protein molecules. In an undefined mechanism requiring Mg, K, and adenine nucleotides, GroEL releases proteins in a form that may progress to the native state, depending on the folding environment (Viitanen et al., 1990). Under conditions where the target protein has been demonstrated not to spontaneously fold (i.e. nonpermissive conditions), the GroES chaperonin is also required (Schmidt et al., 1994). GroEL hydrolyzes ATP even in the absence of unfolded polypeptides; this activity can be inhibited by GroES (Chandrasekhar et al., 1986). The level of GroEL ATPase inhibition by GroES is dependent on the potassium concentration, with greater inhibition seen at lower concentrations of potassium (Todd et al., 1993). The GroES co-chaperonin has been proposed to ``coordinate'' GroEL ATP hydrolysis by binding tightly to one toroid of GroEL, creating an asymmetric complex with ADP trapped at half of the GroEL ATP-binding sites (GroES(7) bullet ADP (7) bullet GroEL(7)-GroEL(7); Todd et al., 1993). This complex can be isolated by gel filtration and dissociates with t of geq5 h. If, however, the isolated complex is allowed to undergo a single round of ATP hydrolysis (12 s), both GroES(7) and ADP are completely exchanged (Todd et al., 1994).

Typical in vitro assays for protein folding involve the addition of denatured target protein to the chaperonin complex, and the appearance of active target protein is followed. Initial formation of the denatured protein is achieved either by using chemical denaturants (urea or guanidinium chloride), heat, or acid. It is generally assumed that the residual concentration of denaturant has no effect on chaperonin activity. Although this is obviously true for dilute concentrations of acid, chemical denaturants have profound effects both on chaperonin activity and on the folding of substrate proteins. Concentrations of guanidinium chloride < 0.1 M, typical of those carried over with denatured proteins, are known to perturb the rate of spontaneous refolding of lactate dehydrogenase (Zettlemeissl et al., 1979). Similarly, we show here that guanidinium chloride increases the rate of aggregation of unfolded conformers of Rubisco, (^1)thereby decreasing the yield of folded protein.

Marked changes in the GroEL tertiary structure (decreased ellipticity, fluorescence, decreased molecular weight) have been observed at guanidinium concentrations in excess of 1.0 M (Price et al., 1993). The ATPase activity was more sensitive, with a 50% reduction in the ATP hydrolysis rate with 300 mM guanidinium chloride (Price et al., 1993). Here we demonstrate that lower concentrations of guanidinium (<150 mM) also inhibit the ATPase of GroEL and destabilize the asymmetric chaperonin complex, thus relieving the inhibition of GroEL ATPase by GroES.


EXPERIMENTAL PROCEDURES

Protein Purification and Assays

Chaperonin proteins from E. coli containing the plasmid pGroESL (Goloubinoff et al., 1989b) were purified as described previously (Todd et al., 1993), ensuring that neither chaperonin had contaminating ATP-utilizing activities. Hydrolysis of [-P]ATP by GroEL was determined using isopropyl acetate extraction of phosphomolybdate complex, as described previously (Todd et al., 1993), using the buffers indicated in the figure legends. Protein determination was by quantitative amino acid analysis. Recombinant dimeric Rubisco was purified from E. coli containing the plasmid pRR2119 (Somerville and Somerville, 1984) encoding the Rubisco gene from Rhodospirillum rubrum as described previously (Pierce and Gutteridge, 1986) and quantitated using the published extinction coefficient ( = 67,200 M cm; Schloss et al., 1982). Rubisco concentrations refer to subunit concentrations of the dimeric enzyme. Rubisco activity was determined by quantitating ^14CO(2) incorporation into acid-stable products, according to Pierce and Gutteridge(1986).

Loss of [alpha-P]ADP from the Asymmetric Chaperonin Complex

The GroES(7) bullet [alpha-P]ADP(7) bullet GroEL(7)-GroEL(7) complex was prepared by incubating 100 µM [alpha-P]ATP (69 Ci/mol) with 2.5 µM GroEL(14) and 5 µM GroES(7) in 50 mM Tris-HCl (pH 7.4), 1 mM KCl, 10 mM Mg(CH(3)CO(2))(2) for 15 min at 25 °C, followed by gel filtration chromatography (PD-10, Pharmacia Biotech Inc.) in 20 mM Tris-HCl (pH 7.4), 10 mM Mg(CH(3)CO(2))(2). The isolated complex contained 5.4-7.4 mol of ADP/mol of GroEL(14) (now at 450 nM). 40 µl of complex was diluted to 160 µl with various solutions to give the desired final salt concentration. Reactions were incubated for various lengths of time in the upper compartment of a micro ultrafiltration device (Amicon, 30-kDa membrane). The amount of released [alpha-P]ADP was quantitated by centrifuging the devices for 30 s at 5000 times g, then counting a small volume of both the top and bottom compartment. Control experiments where a portion of the filtrate was analyzed by gel filtration (TSK 4000) confirmed that neither chaperonin complex (860 kDa) nor GroES(7) (60 kDa) passed through the membrane. Incubating isolated complex with ATP confirmed complete loss of bound [alpha-P]ADP (Todd et al., 1994).

Denatured Protein-induced Dissociation

Solutions of malate dehydrogenase and citrate synthase (Boehringer Mannheim), casein and carboxymethylated alpha-lactalbumin (Sigma), rhodanese (a kind gift of Paul Horowitz), and Rubisco (Pierce and Gutteridge, 1986) were prepared at geq11 mg/ml in 100 mM Tris-HCl (pH 7.8), 10 mM MgCl(2), 1 mM KCl, 0.1 mM EDTA, 10 mM DTT. Chaperonin complex was formed by incubating 2.3 µM GroEL(14) and 5.7 µM GroES(7) with 100 µM [alphaP]ATP (120 Ci/mol) for 10 min in the above buffer. The 500-µl reaction was gel-filtered to remove nontightly bound nucleotide, and the eluent was diluted to 0.52 µM GroEL(14). A 160-µl aliquot was placed in the upper half of a 30-kDa ultrafiltration device (Millipore ultra-free). Denatured protein (10 µl of 11 µM acid-denatured or 2.0 µl of 55 µM protein in either 6 M guanidinium chloride, 100 mM Tris (pH 7.8), 0.1 mM EDTA, or 7.5 M urea, 100 mM Tris (pH 7.8), 0.1 mM EDTA) was rapidly added. After 10 min, samples were centrifuged at 7500 times g for 60 s, resulting in 50% of the liquid in the filtrate, 50% in the retentate. [alphaP]ADP was determined in 2 times 25-µl aliquots of the filtrate. Membrane integrity was confirmed by noting the absence of chaperonin in the filtrate.

Dissociation of the Chaperonin Complex Measured by Release of GroES(7)

Asymmetric chaperonin complex was generated by incubating 5 µM GroEL(14) with 7 µM GroES(7) and 100 µM ATP in 10 mM Tris-HCl (pH 7.4), 10 mM MgCl(2), 0.5 mM KCl, then isolated from excess nucleotide and GroES by gel filtration chromatography (300-mm Bio-Sil Sec-4000, Bio-Rad) in the same buffer. A peak of GroEL(14) and GroES(7) bullet ADP(7) bullet GroEL(7)-GroEL(7) complex elutes at 7 min, and unbound GroES elutes at 9 min. Isolated complex was treated as described, and re-injected. Integration of A allowed quantitation of the amount of GroES released.

Rubisco Folding Reactions

Acid-denatured Rubisco (5 µM in 10 mM HCl) was rapidly diluted 25-fold to 5 ml in GroEL + buffer solution, resulting in a stock of GroEL-bound folding intermediate at 200 nM GroEL(14), 200 nM Rubisco folding intermediate, 300 nM GroES(7) in 40 mM HEPES (pH 7.8), 20 mM DTT, 1.0 mM EDTA, and 0.02% Tween 20 at 0 °C. The stock mixture was warmed to 25 °C, and the folding reaction was begun by diluting 500 µl of the chaperonin/Rubisco folding intermediate 2-fold into an appropriate guanidinium or urea solution containing (final concentrations) 1 mM ATP, 10 mM KCH(3)CO(2), and 10 mM Mg(CH(3)CO(2))(2). Chaperonin-assisted Rubisco folding was quenched by adding 10 µl of 0.1 M glucose, 300 units/ml hexokinase (Sigma, Type C-300) to 100 µl of each folding reaction at the indicated times and measuring Rubisco activity (as described above). The amount of folded Rubisco was fit (using KaleidaGraph) to an exponential decay function, giving the rate of Rubisco folding.

Spontaneous folding was examined by rapidly diluting 20 µl of 5.0 µM acid-denatured Rubisco (in 10 mM HCl) at 25 °C to 1.0 ml with buffer, resulting in a folding reaction containing 100 nM Rubisco folding intermediate, 300 mM NaCl, 100 mM HEPES, 10 mM DTT, 5 mM Mg(CH(3)CO(2))(2), 5 mM KCH(3)CO(2), 0.1 mM EDTA, 5 µM bovine serum albumin, and various concentrations of guanidinium or urea. At the indicated times, 5 µl of 2.86 µM GroEL(14) was added to 100 µl of the reaction, effectively quenching further folding by binding unfolded Rubisco species (U + U in Fig. SII). Samples were then assayed for Rubisco activity.


Scheme II: Scheme II



Aggregation of Rubisco was determined by rapidly diluting Rubisco (5-10 µM in 10 mM HCl at 25 °C) geq 50 times, to 100 nM in 100 mM HEPES (pH 7.8), 10 mM DTT, 0.1 mM EDTA, 5 µM bovine serum albumin, and various concentrations of guanidinium chloride or urea. At the indicated times, the amount of Rubisco folding intermediate which could productively bind GroEL was determined by adding 5 µl of 2.86 µM GroEL(14) to 100 µl of the aggregation reaction. GroEL-bound Rubisco was supplemented with 10 µl of 50 mM Mg(CH(3)CO(2))(2), 50 mM KCH(3)CO(2), 10 mM ATP, 2.5 µM GroES(7) to allow productive folding to occur. Controls lacking this addition confirmed no spontaneous folding occurred in the absence of chloride ion (Schmidt et al., 1994). The residual amount of Rubisco folding intermediate present as a function of time was fit to a pseudo-first order decay function, 1 - exp(-k1times time), using Kaleidagraph.

Light scattering measurements were done on a Perkin-Elmer LS-5B luminescence Spectrometer at 25 °C. Excitation was at 500 nm (10 nm slit), and scattered light was measured by monitoring emission at 500 nm (10 nm slit) for the indicated time.


RESULTS

Guanidinium Chloride Effect on GroEL ATPase Activity

Effect of Guanidinium Chloride on the ATPase of GroEL ± GroES

Price et al.(1993) demonstrated that 300 mM guanidinium chloride inhibits the rate of GroEL ATPase hydrolysis by 50%. Fig. 1examines the effects of lower guanidinium concentrations on ATP hydrolysis at high (A) and low (B) potassium ion concentrations, in the presence and absence of the co-chaperonin GroES. At high concentrations of K, the ATPase activity of GroEL decreased only slightly (20% decrease) upon addition of up to 150 mM guanidinium, whereas the rate of ATP hydrolysis by the GroEL bullet GroES complex (initially 50% of that seen with GroEL alone (Gray and Fersht, 1992; Jackson et al., 1993; Todd et al., 1993)] decreased by 60%. NaCl slightly depressed the ATP hydrolysis rate (20% decrease at 500 mM) and had no effect on GroES inhibition. Thus the influence of guanidinium chloride on GroEL ATPase activity and on its interaction with GroES was not simply an ionic strength effect.


Figure 1: Effect of salts on rate of GroEL ATP hydrolysis. The rate of ATP hydrolysis by GroEL was determined at 25 °C in buffer 1 (100 mM Tris-HCl (pH 7.8), 10 mM MgCl(2), 1 mM DTT, 0.1 mM EDTA) plus the indicated concentrations of freshly prepared guanidinium chloride (circles) or NaCl (squares). Assays were begun by adding [-P]ATP (final concentration = 100 µM, 52 Ci/mol) to reactions containing 0.50 µM GroEL plus 0 (filled symbols) or 1.0 µM GroES (open symbols) and 100 mM (A) or 1 mM (B) KCl. The concentration of liberated P(i) was determined at geq4 times within 15 min. Linear regression analysis (r geq 0.995) gave initial rates of ATP hydrolysis. Note the design of these assays ignores the burst of GroEL ATP hydrolysis seen in the presence of GroES (Todd et al., 1993). Less than 15% of the total amount of ATP present was hydrolyzed under these conditions. Assays at 1 mM KCl included 1 mM creatine phosphate and 1 unit/ml creatine phosphokinase to prevent accumulation of ADP, which is a potent ATPase inhibitor of GroEL bullet GroES at low potassium concentrations (Todd et al., 1993). At 1 mM potassium, less than 10% of the total [P]P(i) present was liberated, thus dilution of specific activity by the ATP-regenerating system was negligible. Rates of asymmetric complex dissociation (asterisk) were calculated from Fig. 2A.




Figure 2: Stability of GroES(7) bullet ADP(7) bullet GroEL(7)-GroEL(7) complex: retention of [alpha-P]ADP. A, percent of initially isolated [alpha-P]ADP which remains associated with the chaperonin complex after 2 min incubation in buffer 2 [10 mM Tris-HCl (pH 7.4), 5 mM Mg(CH(3)CO(2))(2)] plus: guanidinium chloride (), NaCl (bullet), Tris-HCl (pH 7.8) (times), or Mg(CH(3)CO(2))(2) (). Complex dissociation was also measured in 10 mM Tris-HCl (pH 7.4), 50 mM Mg (CH(3)CO(2))(2) with increasing guanidinium chloride (box), or NaCl (circle). B, percent of initially isolated [alpha-P]ADP which remains associated with the chaperonin complex after incubation in buffer 2 with 0 () or 80 (bullet) mM guanidinium chloride.



At low potassium concentration, the affinity of GroEL for ATP is much reduced (Todd et al., 1993), and the effect of guanidinium is more pronounced, with an 80% decrease in activity upon addition of 150 mM (Fig. 1B). A portion of this inhibition could be reproduced by increasing ionic strength alone, as evidenced by a 50% decrease in ATPase activity in the presence of NaCl. Fig. 1B also emphasizes the specificity of GroEL for K in the presence of several hundredfold excess of Na. The inhibition of GroEL by GroES at low potassium concentrations is nearly complete and occurs after one GroEL toroid has turned over (Todd et al., 1993). Guanidinium chloride (geq60 mM) relieved this inhibition, and ATP hydrolysis became independent of GroES. The similarity in rates of ATP hydrolysis regardless of GroES indicated that guanidinium-induced dissociation of the complex became faster than the rate of ATP turnover by GroEL alone. Elevated concentration of NaCl alone, however, could also relieve GroES inhibition, suggesting that the GroEL bullet GroES bullet ADP complex is unstable at higher ionic strengths.

Dissociation of the Chaperonin Complex in Guanidinium Chloride: Loss of ADP

The stable asymmetric chaperonin complex (GroES(7) bullet ADP(7) bullet GroEL(7)-GroEL(7)) can be isolated by gel-filtration chromatography, and the bound nucleotide dissociates with tof 5 h at 25 °C (Todd et al., 1993, 1994). During steady-state ATP hydrolysis, however, the ADP is lost after each toroid turns over. Measuring retained [alpha-P]ADP, therefore, becomes an extremely sensitive method of determining complex stability. Fig. 2A demonstrates the amount of nucleotide retained in the complex after a 3-min incubation with various salts. Increasing buffer/salt concentrations destabilized the complex, such that at 500 mM, 50% of the complex dissociated within 3 min. The complex was much more sensitive to guanidinium, however, with >50% dissociation seen above 60 mM guanidinium chloride. The guanidinium-induced dissociation curve could be shifted to lower concentration by including other salts (data not shown) or to higher concentrations by raising the Mg concentration. Mg also slowed dissociation seen at high concentrations of other salts. GroEL tertiary structure has previously been shown to be Mg-sensitive (Azem et al., 1994). Varying the pH from 7.0 to 9.0 did not affect complex stability.

The rate of nucleotide loss (Fig. 2B) in 80 mM guanidinium was 3.1 min, a 100-fold increase in the dissociation rate. At any given guanidinium concentration, the dissociation rate was decreased by the presence of additional ADP, (^2)consistent with ADP binding on the second ring of GroEL stabilizing the chaperonin complex. Similar effects of ADP on complex stability have been demonstrated at 0 °C (Todd et al., 1994).

Dissociation of the Chaperonin Complex in Guanidinium Chloride: Loss of GroES

We have previously suggested that the dissociation of ADP and GroES from the asymmetric chaperonin complex is an ordered process; GroES dissociates before the bound ADP (Todd et al., 1994). Therefore, guanidinium-induced loss of the tightly bound ADP must imply that GroES has also dissociated. GroES dissociation was monitored by gel-filtration chromatography (Fig. 3). Asymmetric chaperonin complex was formed, then separated from excess ligands. Re-isolation of the chaperonin complex after 15 or 75 min demonstrated only 3 and 14%, respectively, of the total GroES bound had dissociated. After 5-min treatment with 80 mM guanidinium, however, 67% of the total bound GroES had dissociated. The dissociation rates calculated from this technique were in agreement with those based on the rate of ADP loss (Fig. 2B) and confirmed that in the presence of guanidinium both tightly bound GroEL ligands rapidly dissociated.


Figure 3: Dissociation of GroES from asymmetric complex as measured by gel filtration. Isolated asymmetric chaperonin complex was injected onto a TSK 4000 after: (i) 15-min, (ii) 75-min, and (iii) 5-min treatment with 80 mM guanidinium chloride or (iv) 5-min incubation with 20 mM EDTA. The GroEL and chaperonin complex elutes at 6.9 ml (Peak A), and GroES elutes at 9.3 ml (Peak B). Absorbance at 212 nm was monitored and is shown offset by 70, 60, and 30 multiabsorbance units for runs i, ii, and iii, respectively.



The Effect of Unfolded Protein and Protein Denaturants on the Chaperonin Stability

Unfolded proteins have been suggested to play an active role in the chaperonin ATPase cycle by inducing the dissociation of the asymmetric complex (Martin et al., 1993). Given the instability of the complex to guanidinium (Fig. 1-3), we measured dissociation upon addition of denatured proteins, using acid, 8 M urea, or 6 M guanidinium chloride as denaturants (Table 1). Two proteins purported to exist in stable, unfolded states (reduced, carboxymethylated alpha-lactalbumin and casein; Hayer-Hartl et al., 1994; Martin et al., 1991) were also included. None of the six unfolded proteins induced the release of tightly bound [alphaP]ADP, including two which have previously been reported to do so (Martin et al., 1993). Accelerated release of the bound [alphaP]ADP was only observed in the presence of guanidinium.



Guanidinium Effects on Rubisco Folding

Since in vitro assays of chaperonins typically involve guanidinium or urea-denatured proteins, we examined the effects of increasing concentrations of denaturants on the rate and extent of chaperonin-mediated protein folding. Fig. 4A depicts the extent to which acid-denatured Rubisco had refolded at various times; first-order rate constants and final yields are reported in Table 2. Guanidinium, but not urea, decreased both the rate and the yield of folded protein. The decrease in folding rate was caused by slowing the chaperonin ATPase cycle (Fig. 1A), whereas the decrease in folding yield was caused by an increase in the rate of Rubisco aggregation (see below).


Figure 4: Effect of guanidinium chloride on Rubisco folding. A, chaperonin-assisted Rubisco folding in the presence of 0 (), 50 (), 100 (asterisk), 150 (up triangle), or 200 (+) mM guanidinium chloride. B, spontaneous folding of acid-denatured Rubisco was measured in the presence of 0 (), 30 (bullet), 60 (), or 90 (asterisk) mM guanidinium chloride or 200 mM urea ().





We further examined the effect of denaturants on the spontaneous folding reaction at 25 °C (Fig. 4B, Table 2). Spontaneous folding of R. rubrum Rubisco at 25 °C is dependent on chloride ions (Lorimer et al., 1993). Addition of guanidinium chloride had no effect on the rate of spontaneous folding, yet the yield of folded protein decreased considerably (Table 2), even though additional chloride ion was being added. Urea, on the other hand, slightly reduced both the rate and the yield of Rubisco folding. A reduced yield, with no effect on the rate-limiting step of folding, suggested that the partitioning between productive and unproductive folding pathways had changed; possibly through an increase in the formation of species which do not fold to the native state. This hypothesis was first tested by measuring the rate at which Rubisco conformer(s) became unrecognizable by GroEL. Unfolded Rubisco conformer(s) were created by diluting acid-denatured protein into buffer-containing denaturants. In the absence of chloride ions, these Rubisco conformers do not spontaneously fold (Lorimer et al., 1993; Schmidt et al., 1994), and light scattering demonstrated that these species do not form large aggregates (see below). Excess GroEL was added at various times to bind these Rubisco conformers, and the amount of trapped Rubisco was measured. Guanidinium (but not urea) increased the rate at which these conformer(s) became unrecognizable to GroEL in a concentration-dependent manner, almost tripling the rate (Table 2). Urea slightly slowed this rate.

The simplest interpretation for the disappearance of unfolded conformers (other than progressing to the native state) is that these species aggregate. Chaperonins are known to suppress formation of high molecular weight aggregates which can easily be detected using light-scattering techniques (Buchner et al., 1991). Therefore we tested guanidinium for its effect on the rate of Rubisco aggregation. In the absence of guanidinium, neutralization of acid-denatured Rubisco resulted in a species which did not scatter light for at least 15 min (Fig. 5B). Upon addition of guanidinium HCl (but not urea), the rate and extent of light scattering increased. The increased scattering was not simply due to increased ionic strength, since addition of up to 250 mM potassium acetate failed to demonstrate a corresponding increase in aggregation. Rather, guanidinium allowed growth of Rubisco species, no longer recognizable by GroEL into particles large enough to scatter light.


Figure 5: Effect of guanidinium chloride on the rate of Rubisco aggregation. A, acid-denatured Rubisco was added to buffers containing 0 (), 30 (bullet), 60 (), or 90 (asterisk) mM guanidinium chloride or 200 mM urea (). GroEL was added at the indicated time to trap any Rubisco folding intermediate which had not progressed irreversibly to unproductively folded states. The trapped Rubisco was subsequently released and allowed to fold. B, light scattering of unfolded Rubisco (added at 30 s, first arrow) was monitored for 15 min (trace a). After 300 s (second arrow), 60 (trace b), 90 (trace c), 120 (trace d) mM guanidinium chloride, or 200 (trace e) mM urea was added.




DISCUSSION

Effect of Unfolded Protein on the Stability of the Chaperonin Complex

The GroEL chaperonin forms a stable complex with the GroES co-chaperonin, which contains tightly bound nucleotide. A model for chaperonin-assisted protein folding was presented (Martin et al., 1993) in which the binding of an unfolded substrate protein induced the dissociation of the chaperonin complex. Table 1reveals that none of the examined unfolded proteins could induce complex dissociation unless they were denatured in guanidinium chloride. Guanidinium alone, however, was sufficient to cause dissociation, even in the absence of unfolded proteins (Fig. 2). Therefore a key element in the model of Martin et al.(1993), namely nucleotide exchange, specifically induced by unfolded protein is probably not due to unfolded proteins but to the residual guanidinium with which they were delivered. Physiologically the release of the tightly bound ADP occurs only as a consequence of ATP hydrolysis on the unliganded GroEL toroid.

Effect of Guanidinium Chloride on Chaperonin Activity

GroEL structure has previously been studied in the presence of elevated concentrations of denaturants: Lissin et al.(1990) used geq4 M urea, and Price et al.(1993) used geq1.5 M guanidinium chloride. Both studies demonstrated formation of monomeric species and complete loss of secondary structure at high denaturant concentrations. Regain of secondary structure was achieved upon removal of the denaturant; however, only when the chaperone had been denatured with urea was a regain in functionality demonstrated (Lissin et al., 1990). Mizobata and Kawata(1994) have recently extended these studies and found that even at 0.5 M guanidinium chloride, GroEL demonstrated measurable changes in the fluorescence spectrum and in 1-anilino-8-naphthalenesulfonate dye binding. The ATPase activity of GroEL was more sensitive to guanidinium ion: at 300 mM guanidinium, the ATPase was 50% inhibited (Price et al., 1993), a value we have confirmed at high potassium concentrations (Fig. 1A). In the presence of GroES, however, ATP hydrolysis was reduced by half at 60 mM guanidinium due to decreased affinity of the unliganded sites on GroEL for ATP. Thus under nonpermissive conditions (where GroES is required for folding) the residual effects of guanidinium on the chaperonins were more pronounced.

Fig. SIsummarizes the ATPase cycle of the GroEL double-toroid (as viewed from the side, species A; GroEL(7)-GroEL(7)). In the absence of GroES, hydrolysis proceeds through Cycle I, with the rate of ATP hydrolysis being slower than either ATP binding or P(i) release (Jackson et al., 1993; Todd et al., 1993). In the presence of GroES, the reaction cycle involves the formation of an asymmetric complex (GroES(7) bullet ADP(7) bullet GroEL(7)-GroEL(7)), which subsequently breaks down, then reforms (either through species B D F A, or through species B D E C). At low potassium concentrations, the affinity of the unliganded toroid of GroEL for ATP is greatly reduced (Todd et al., 1993), and species B is stable and can be isolated. The affinity for ATP can be subsequently increased by raising the concentration of K and ATP hydrolysis restored. With each turnover on the free face of GroEL, the tightly bound ADP on the opposite toroid is completely exchanged (Todd et al., 1994). If all seven nucleotide binding sites on the unliganded toroid must be simultaneously occupied for quantized turnover to occur (Todd et al., 1994), a reduced affinity for ATP at each binding site decreases the probability of complete occupancy and hence the frequency of ATP hydrolysis by that ring. Thus at low potassium concentrations the affinity for ATP is reduced to the extent that species D is never completely occupied, and the asymmetric complex B remains completely inhibited. However, increases in the ionic strength in general, and increases in the concentration of guanidinium HCl in particular restored ATP hydrolysis (Fig. 1B). The restoration of ATP hydrolysis by species B requires either (i) that the affinity of the unliganded GroEL ring for ATP be increased so as to populate species D or (ii) that GroES dissociates from species B, thus returning the system to the uninhibited Cycle I. Since we have demonstrated that the rate of GroES dissociation is increased >100-fold in the presence of 80 mM guanidinium HCl ( Fig. 2and Fig. 3), we favor the latter explanation. At low potassium concentrations, an increase in ionic strength or in the concentration of guanidinium HCl causes the chaperonin complex to dissociate and return to cycle I (i.e. species B F A). The rate of the dissociation step in various concentrations of guanidinium was calculated from the data in Fig. 2and is shown in Fig. 1B (asterisk). At concentrations in excess of 100 mM, the complex dissociated faster than it could be formed, thus ATP hydrolysis became independent of GroES. At higher potassium concentrations, the asymmetric complex dissociation may remain rate-limiting, or the affinity for ATP may be sufficiently high to permit formation of species D. We cannot presently distinguish these two alternatives.


Scheme I: Scheme I



The addition of guanidinium chloride to a fully inhibited chaperonin complex temporarily relieves GroES inhibition, allowing hydrolysis of additional ATP. Under such circumstances GroEL continues hydrolyzing ATP until the ADP concentration again rises to inhibitory levels (Todd et al., 1993). The addition of guanidinium chloride thus induces a ``burst'' of ATP hydrolysis. The magnitude of this burst will depend on the the ionic strength and on the concentrations of K, guanidinium chloride, ATP, and ADP. Martin et al.(1991) observed just such a burst upon adding rhodanese, unfolded in guanidinium chloride, to a fully inhibited chaperonin complex. They attributed this phenomenon to a specific stimulation of the chaperonin ATPase activity. In view of the inability of unfolded proteins, denatured by other methods, to induce the dissociation of the chaperonin complex (Table 1), plus the ability of even modest concentrations of guanidinium chloride alone to induce bursts of ATP hydrolysis, we question the interpretation of Martin et al. (1991).

Guanidinium Effects on Protein Folding Reactions

We have previously suggested that chaperonin-assisted folding occurs by an iterative annealing mechanism (Todd et al. 1994). Folding is determined by kinetic partitioning () between species which can progress to the native state (N) and an ensemble of conformers which become kinetically trapped (U; Fig. SII). These trapped conformers bind to the chaperonin, are released in an less folded state (U) and partition to N or U again and again. The appearance of folded protein depends upon: 1) the rate constant for the rate-limiting folding step(s) (k(f)); and 2) the steady-state concentration of the uncommitted protein (U) released from GroEL (i.e. apparent folding rate = d[N]/dt = k(f) bullet [U]). The steady-state concentration of U, in turn, depends upon the rate of the partitioning reactions, the rate of release of U from chaperonin (k(d)), and the kinetic/thermodynamic barriers involved in reversing the reaction leading to U (k). The effect of slowing the ATP hydrolysis rate on the folding rate will depend on whether k(d) > k or k(d) < k. When k(d) > k, a decrease in the rate of ATP hydrolysis (and therefore the rate of peptide release) will decrease the concentration of U, thus slowing the folding rate. If misfolded structures can readily revert to U, k(d) < k, the protein folds spontaneously, and slowing the rate of ATP hydrolysis will have no effect on the folding rate. For Rubisco folding, since a decrease in the rate of ATP hydrolysis is accompanied by a decrease in the rate of chaperonin-assisted folding, it follows that k(d) > k. This conclusion is also consistent with our recent observation that the rate of chaperonin-assisted folding of Rubisco is proportional to the chaperonin concentration. (^3)

Spontaneous folding experiments demonstrated that guanidinium decreased the yield, but had no effect on the rate of folding. Instead, guanidinium increased the rate of aggregate formation (k) (Fig. 5), decreasing the amount of protein available for folding (U + U). The chaperonin-assisted folding reaction was less susceptible to aggregation, since GroEL could re-bind the misfolded conformers and allow re-partitioning to occur.

GroEL has now been observed to increase the rate of folding for two proteins: Rubisco (4-fold in Table 2; Lorimer et al., 1993) and malate dehydrogenase (Staniforth et al., 1994). This rate increase could occur simply by increasing the concentration of the species which has the potential to fold (Fig. SII, species U). An increase in the rate constant, however, indicates that there is a change in the rate-determining step, perhaps from k to k(d). In agreement with this hypothesis, Sosnick et al.(1994) demonstrated that the spontaneous folding rate for the majority of cytochrome c conformers is limited by the reversal of misfolded structures (k). For many proteins GroEL decreases the rate of folding, perhaps by lowering the concentration of U by binding U. A net decrease in protein available for folding results when k(d) < k, an increase occurs when k(d) > k.

While many folding characteristics are specific to the protein being studied, the difficulties encountered by using guanidinium could be more universal. In comparing the folding yields of several proteins (Table 3), higher yields of spontaneous folding occurred when proteins were denatured in urea than when guanidinium chloride was used. Given the relatively slow mixing which occurs upon dilution of these viscous solutions (seconds), and thus the high local concentration of both denaturant and unfolded polypeptide, it is possible that enhanced intermolecular hydrophobic associations result in increased aggregation. Many in vitro protein folding reactions are performed by diluting a small volume of highly concentrated denatured protein by a large factor resulting in some variability in the final denaturant concentration. Given the sensitivity of the chaperonin interactions to low concentrations of guanidinium chloride and its possible effects on unfolded proteins, we suggest that denaturants other than guanidinium chloride be used when assessing chaperone-assisted folding activity.




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. Tel.: 302-695-4584; Fax: 302-695-4509.

(^1)
The abbreviations used are: Rubisco, ribulose-1,5-bisphosphate carboxylase; DTT, dithiothreitol.

(^2)
M. J. Todd and G. H. Lorimer, unpublished results.

(^3)
M. J. Todd and G. H. Lorimer, submitted for publication.


ACKNOWLEDGEMENTS

We thank Tony Gatenby and Paul Viitanen for critically reading this manuscript and Tom Webb for technical assistance.


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