P but not R-axis interface is involved in cooperative binding of NAD on tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Homotetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three non-equivalent P, R, and Q interfaces. In our previous study, negative cooperativity in NAD binding to wild-type GAPDH was interpreted according to the induced-fit model in terms of two independent dimers with two interacting binding sites in each dimer. Two dimeric mutant GAPDHs, i.e. Y46G/S48G and D186G/E276G, were shown to exhibit positive cooperativity in NAD binding. Based on the molecular modeling of the substitutions and the fact that the most extensive inter-subunit interactions are formed across the P-axis interface of the tetramer, it was postulated that both dimeric mutant GAPDHs were of O-P type. Therefore, the P-axis interface was assumed to play a major role in causing cooperativity in NAD binding.Here, two other mutant GAPDHs, Y46G/R52G and D282G, have been studied. Using small angle X-ray scattering, the dimeric form of the D282G mutant GAPDH is shown to be of O-R type whereas both dimeric mutant GAPDHs Y46G/R52G and Y46G/S48G are of O-P type. Similarly to dimeric Y46G/S48G mutant GAPDH, the dimeric Y46G/R52G mutant GAPDH exhibits positive cooperativity in NAD binding. On the other hand, no significant cooperativity in NAD binding to the dimeric form of the D282G mutant GAPDH is observed, whereas its tetrameric counterpart exhibits negative cooperativity, similarly to the wild-type enzyme. Altogether, the results support the view that the P-axis interface is essential in causing cooperativity in NAD binding by transmitting the structural information induced upon cofactor binding from one subunit to the other one within O-P/Q-R dimers in contrast to the R-axis interface, which does not transmit structural information within O-R/Q-P dimers. The absence of activity of O-P and O-R dimer GAPDHs is the consequence of a pertubation of the conformation of the active site, at least of the nicotinamide subsite, as evidenced by the absence of an ion pair between catalytic residues C149 and H176 and the greater accessibility of C149 to a thiol kinetic probe.     

Inhibition of chaperonin GroEL by a monomer of ovine prion protein and its oligomeric forms

The possibility of inhibition of chaperonin functional activity by amyloid proteins was studied. It was found that the ovine prion protein PrP as well as its oligomeric and fibrillar forms are capable of binding with the chaperonin GroEL. Besides, GroEL was shown to promote amyloid aggregation of the monomeric and oligomeric PrP as well as PrP fibrils. The monomeric PrP was shown to inhibit the GroEL-assisted reactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The oligomers of PrP decelerate the GroEL-assisted reactivation of GAPDH, and PrP fibrils did not affect this process. The chaperonin GroEL is capable of interacting with GAPDH and different PrP forms simultaneously. A possible role of the inhibition of chaperonins by amyloid proteins in the misfolding of the enzymes involved in cell metabolism and in progression of neurodegenerative diseases of amyloid nature is discussed.     

GroEL-assisted dehydrogenase folding mediated by coenzyme is ATP-independent.

It has been commonly accepted that GroEL functions as a chaperone by modulation of its affinity for folding intermediates through binding and hydrolysis of ATP. However, we have found that NAD, as a coenzyme of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), also stimulates the discharge of GAPDH folding intermediate from its stable complex with GroEL formed in the absence of ATP and assists refolding with the same yield as ATP/Mg(2+) does. The reactivation further increases when ATP is also present, but addition of Mg(2+) has no more effect. NADP, a coenzyme of glucose-6-phosphate dehydrogenase, also releases its folding intermediates from GroEL and increases reactivation. Different from ATP, NAD triggers the release of GAPDH intermediates bound by GroEL via binding with GAPDH itself but not with GroEL, and the released intermediates all folded to native molecules without the formation of aggregation. The collaborative effects of coenzyme and GroEL mediate GroEL-assisted dehydrogenase folding in an ATP-independent way.     

Participation of chaperonin GroEL in the folding of D-glyceraldehyde-3-phosphate dehydrogenase. An approach based on the use of different oligomeric forms of the enzyme immobilized on sepharose

The binding of denatured B. stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the E. coli chaperonin GroEL was investigated in two systems: (1) GroEL immobilized on Sepharose via a single subunit was titrated with urea-denatured soluble GAPDH and (2) a Sepharose-bound denatured GAPDH monomer was titrated with soluble GroEL. Similar apparent K _D values for the complex GroEL·GAPDH were obtained in both cases (0.04 and 0.03 M, respectively), the stoichiometry being 1.0 mol chaperonin per GAPDH subunit in the system with the immobilized GroEL and 0.2 mol chaperonin per Sepharose-bound GAPDH monomer. Addition of GroEL and Mg·ATP to a reactivation mixture increased the yield of reactivation of both E. coli and B. stearothermophilus GAPDHs. Incubation of the Sepharose-bound catalytically active tetrameric and dimeric GAPDH forms with the protein fraction of a wild-type E. coli cell extract resulted in the binding of GroEL to the dimer and no interaction with the tetrameric form. These data suggest that GroEL may be capable of interacting with the interdimeric contact regions of the folded GAPDH dimers.     

Thermal unfolding used as a probe to characterize the intra- and intersubunit stabilizing interactions in phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three nonequivalent interfaces. To investigate the contribution of intra- and intersubunit interactions to GAPDH thermostability, 10 residues located either at the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic domain (amino acids 149-312) were mutated and the thermal unfolding of the mutants was studied by differential scanning calorimetry in the absence and presence of saturating concentrations of NAD. Disruptions of intrasubunit interactions lead to a drastic decrease in thermostability of the N313T, Y283V, and W310F mutants. Moreover, for the N313T mutant, a weakening of cooperative interactions between the catalytic and the cofactor domains and an inefficient binding of NAD are observed. This is likely the consequences of modification or loss of the hydrogen bonding network associating N313 and residues 236-238 and N313 and the nicotinamide carboxyamide of NAD, respectively. For the residues Y283 and W310, which are involved in stacking hydrophobic interactions, mutating both positions does not affect the efficiency of NAD binding. This shows that the factors involved in the thermostability of the tetrameric apo GAPDH are then different from those induced by NAD binding. Disruption of intersubunit hydrogen bonds between the catalytic domain and the NAD-binding domain of a neighboring subunit also leads to a significant destabilization of the apo tetrameric form as observed for the D282G mutant. Moreover, no efficient binding of NAD is observed. Both results are likely the consequence of a loss of hydrogen bonds across the P-axis and the Q-axis between D282 and R197 and between D282 and R52, respectively. Similar results, i.e., a destabilizing effect and inefficient NAD binding, are observed with the T34Q/T39S/L43Q mutant in which steric hindrance is introduced at the S-loop of the R-axis-related subunit via mutations at the adenosine subsite. The dimeric form of the D282G mutant exhibits a single partial heat absorption peak, whereas the Y46G/R52G mutant which exists only as a dimer shows two peaks. Taking into account the recent small-angle X-ray scattering studies which suggested that the dimeric form of the D282G mutant and of the dimeric Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette, unpublished results), we propose that the presence of one or two peaks in thermal unfolding of dimers is a signature of the dimer type.     

The intermediate domain defines broad nucleotide selectivity for protein folding in Chlamydophila GroEL1

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln(400) and Ile(404) in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.     

Interaction of GroEL and GroEL/GroES complexes with a nonnative subtilisin variant: a small-angle neutron scattering study

Small-angle neutron scattering and contrast variation were used to study the solution structure of GroEL and GroEL/GroES chaperonins complexed with a nonnative variant of the polypeptide substrate, subtilisin (PJ9). The subtilisin was 86% deuterated (dPJ9) so that it contrasted sufficiently with the chaperonin, allowing the contrast variation technique to be used to separate the scattering from the two components bound in the complex. Both the native double-ring GroEL and a single-ring mutant were used with dPJ9 bound in a 1:1 stoichiometry per GroEL toroid. This allowed both the position and the shape of dPJ9 in the GroEL/dPJ9 complexes to be determined. A single-ring GroEL/GroES variant complexed with one dPJ9 molecule was used to study the structural changes of dPJ9 in GroEL/GroES/dPJ9 complexes formed with ADP and with ATP. It was found that both the shape and the position of the bound dPJ9 in the GroEL/GroES/dPJ9 complex with ADP were the same as those in the GroEL/dPJ9 complex. However, dPJ9 assumed a more symmetric shape when bound in the GroEL/GroES/dPJ9 complex with ATP. This important observation reflects the relative ability of ATP to promote refolding of protein substrates relative to that of ADP.     

"Half of the sites" binding of D-glyceraldehyde-3-phosphate dehydrogenase folding intermediate with GroEL

Two D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) folding intermediate subunits bind with chaperonin 60 (GroEL) to form a stable complex, which can no longer bind with additional GAPDH intermediate subunits, but does bind with one more lysozyme folding intermediate or one chaperonin 10 (GroES) molecule, suggesting that the two GAPDH subunits bind at one end of the GroEL molecule displaying a "half of the sites" binding profile. For lysozyme, GroEL binds with either one or two folding intermediates to form a stable 1:1 or 1:2 complex with one substrate on each end of the GroEL double ring for the latter. The 1:1 complex of GroEL.GroES binds with one lysozyme or one dimeric GAPDH folding intermediate to form a stable ternary complex. Both complexes of GroEL.lysozyme1 and GroEL.GAPDH2 bind with one GroES molecule only at the other end of the GroEL molecule forming a trans ternary complex. According to the stoichiometry of GroEL binding with the GAPDH folding intermediate and the formation of ternary complexes containing GroEL.GAPDH2, it is suggested that the folding intermediate of GAPDH binds, very likely in the dimeric form, with GroEL at one end only.     

Catalysis, commitment and encapsulation during GroE-mediated folding.

The Escherichia coli GroE chaperones assist protein folding under conditions where no spontaneous folding occurs. To achieve this, the cooperation of GroEL and GroES, the two protein components of the chaperone system, is an essential requirement. While in many cases GroE simply suppresses unspecific aggregation of non-native proteins by encapsulation, there are examples where folding is accelerated by GroE.Using maltose-binding protein (MBP) as a substrate for GroE, it had been possible to define basic requirements for catalysis of folding. Here, we have analyzed key steps in the interaction of GroE and the MBP mutant Y283D during catalyzed folding. In addition to high temperature, high ionic strength was shown to be a restrictive condition for MBP Y283D folding. In both cases, the complete GroE system (GroEL, GroES and ATP) compensates the deceleration of MBP Y283D folding. Combining kinetic folding experiments and electron microscopy of GroE particles, we demonstrate that at elevated temperatures, symmetrical GroE particles with GroES bound to both ends of the GroEL cylinder play an important role in the efficient catalysis of MBP Y283D refolding. In principle, MBP Y283D folding can be catalyzed during one encapsulation cycle. However, because the commitment to reach the native state is low after only one cycle of ATP hydrolysis, several interaction cycles are required for catalyzed folding.     

GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle

GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.     

Refolding of target proteins from a "rigid" mutant chaperonin demonstrates a minimal mechanism of chaperonin binding and release

One of the most interesting facets of GroEL-facilitated protein folding lies in the fact that the requirement for a successful folding reaction of a given protein target depends upon the refolding conditions used. In this report, we utilize a mutant of GroEL (GroEL T89W) whose domain movements have been drastically restricted, producing a chaperonin that is incapable of utilizing the conventional cyclic mechanism of chaperonin action. This mutant was, however, still capable of improving the refolding yield of lactate dehydrogenase in the absence of both GroES and ATP hydrolysis. A very rapid interconversion of conformations was detected in the mutant immediately after ATP binding, and this interconversion was inferred to form part of the target release mechanism in this mutant. The possibility exists that some target proteins, although dependent on GroEL for improved refolding yields, are capable of refolding successfully by utilizing only portions of the entire mechanism provided by the chaperonins.     

On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding.

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding. The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes. The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial. It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL. By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate. Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding. In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes. These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism.     

Binding of a chaperonin to the folding intermediates of lactate dehydrogenase.

When Bacillus stearothermophilus LDH dimer is incubated with increasing concentrations of the denaturant guanidinium chloride, three distinct unfolded states of the molecule are observed at equilibrium [Smith, C. J., et al. (1991) Biochemistry 30, 1028-1036]. The kinetics of LDH refolding are consistent with an unbranched progression through these states. The Escherichia coli chaperonin, GroEL, binds with high affinity to the completely denatured form and more weakly to the earliest folding intermediate, thus retarding the refolding process. A later structurally defined folding intermediate, corresponding to a molten globule form, is not bound by GroEL; neither is the inactive monomer. The complex between GroEL and denatured LDH is destabilized by the binding of magnesium/ATP (Mg/ATP) or by the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP). From our initial kinetic data, we propose that GroEL exists in two interconvertible forms, one of which is stabilized by the binding of Mg/ATP but associates weakly with the unfolded protein. The other is destabilized by Mg/ATP and associates strongly with unfolded LDH. The relevance of these findings to the role of GroEL in vivo is discussed.     

Non-functioning chaperonin GroEL stimulates protein aggregation

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be decreased in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The size of individual proteins (GroEL, GAPDH, and the recombinant prion) and aggregates formed after their mixing have been determined by the dynamic light scattering analysis. It was shown that at 25°C, the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the size of the aggregates to 70–80 nm. After blocking of one of the chaperonin active sites by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.     

Unfolded, oxidized, and thermoinactivated forms of glyceraldehyde-3-phosphate dehydrogenase interact with the chaperonin GroEL in different ways

The interaction of GroEL with different denatured forms of glyceraldehyde-3-phosphate dehydrogenase* (GAPDH) has been investigated. GroEL does not prevent thermal denaturation of GAPDH, but effectively interacts with the thermodenatured enzyme, thus preventing the aggregation of denatured molecules. Binding of the thermodenatured GAPDH shifts the Tm value of the GroEL thermodenaturation curve by 3 degrees towards higher temperatures and increases the DeltaHcal value 1.44-fold, indicating a significant increase in the thermal stability of the resulting complex. GAPDH thermodenatured in the presence of GroEL cannot be reactivated by the addition of GroES, Mg2+, and ATP. In contrast, GAPDH denatured in guanidine hydrochloride (GAPDHden) is reactivated in the presence of GroEL, GroES, Mg2+, and ATP, yielding 11-15% of its original activity, while the spontaneous reactivation yields only 2-3%. The oxidation of GAPDH with hydrogen peroxide in the presence of 4 M guanidine hydrochloride results in the formation of the enzyme (GAPDHox) that cannot acquire its native conformation and binds to GroEL irreversibly. Binding of GAPDHox to one of the GroEL rings completely inhibits the GroEL-assisted reactivation of GAPDHden, but does not affect the GroEL-assisted reactivation of lactate dehydrogenase (LDH). The data suggest that LDH can be successfully reactivated due to the binding of the denatured molecules to the apical domain of the opposite GroEL ring with their subsequent release into the solution without encapsulation (trans-mechanism). In contrast, GAPDH requires the hydrophilic cavity for the reactivation (cis-mechanism).     

Combined thermodynamic and kinetic analysis of GroEL interacting with CXCR4 transmembrane peptides

GroEL along with ATP and its co-chaperonin GroES has been demonstrated to significantly enhance the folding of newly translated G-protein-coupled receptors (GPCRs). This work extends the previous studies to explore the guest capture and release processes in GroEL-assisted folding of GPCRs, by the reduced approach of employing CXCR4 transmembrane peptides as model substrates. Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven. It is found that the peptides interact with the paired α helices in the apical domain of the chaperonin which are similar with the binding sites of SBP (strongly binding peptide: SWMTTPWGFLHP). Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition. Meanwhile, the binding kinetics of CXCR4 peptides with GroEL was also examined; ATP alone, or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin. The results obtained would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.     

Synchronized domain-opening motion of GroEL is essential for communication between the two rings

Escherichia coli chaperonin GroEL consists of two stacked rings of seven identical subunits each. Accompanying binding of ATP and GroES to one ring of GroEL, that ring undergoes a large en bloc domain movement, in which the apical domain twists upward and outward. A mutant GroEL(AEX) (C138S,C458S,C519S,D83C,K327C) in the oxidized form is locked in a closed conformation by an interdomain disulfide cross-link and cannot hydrolyze ATP (Murai, N., Makino, Y., and Yoshida, M. (1996) J. Biol. Chem. 271, 28229-28234). By reconstitution of GroEL complex from subunits of both wild-type GroEL and oxidized GroEL(AEX), hybrid GroEL complexes containing various numbers of oxidized GroEL(AEX) subunits were prepared. ATPase activity of the hybrid GroEL containing one or two oxidized GroEL(AEX) subunits per ring was about 70% higher than that of wild-type GroEL. Based on the detailed analysis of the ATPase activity, we concluded that inter-ring negative cooperativity was lost in the hybrid GroEL, indicating that synchronized opening of the subunits in one ring is necessary for the negative cooperativity. Indeed, hybrid GroEL complex reconstituted from subunits of wild-type and GroEL mutant (D398A), which is ATPase-deficient but can undergo domain opening motion, retained the negative cooperativity of ATPase. In contrast, the ability of GroEL to assist protein folding was impaired by the presence of a single oxidized GroEL(AEX) subunit in a ring. Taken together, cooperative conformational transitions in GroEL rings ensure the functional communication between the two rings of GroEL.     

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.     

Functional communications between the apical and equatorial domains of GroEL through the intermediate domain

The Escherichia coli GroEL subunit consists of three domains with distinct functional roles. To understand the role of each of the three domains, the effects of mutating a single residue in each domain (Y203C at the apical, T89W at the equatorial, and C138W at the intermediate domain) were studied in detail, using three different enzymes (enolase, lactate dehydrogenase, and rhodanese) as refolding substrates. By analyzing the effects of each mutation, a transfer of signals was detected between the apical domain and the equatorial domain. A signal initiated by the equatorial domain triggers the release of polypeptide from the apical domain. This trigger was independent of nucleotide hydrolysis, as demonstrated using an ATPase-deficient mutant, and, also, the conditions for successful release of polypeptide could be modified by a mutation in the apical domain, suggesting that the polypeptide release mechanism of GroEL is governed by chaperonin-target affinities. Interestingly, a reciprocal signal from the apical domain was suggested to occur, which triggered nucleotide hydrolysis in the equatorial domain. This signal was disrupted by a mutation in the intermediate domain to create a novel ternary complex in which GroES and refolding protein are simultaneously bound in a stable ternary complex devoid of ATPase activity. These results point to a multitude of signals which govern the overall chaperonin mechanism.     

Polyols induce ATP-independent folding of GroEL-bound bacterial glutamine synthetase.

We have previously assessed the GroE chaperonin requirements for folding of bacterial glutamine synthetase (GS) and established that, at 37 degrees C in 50 mM Tris buffer, ATP binding to the GroEL-GS complex is mandatory for the release and reactivation of dodecameric enzyme. However, we demonstrate here that the addition of 1-4 M glycerol to GroEL-GS complexes resulted in release and reactivation of GS in the absence of nucleotide. Furthermore, the kinetics of refolding and refolding yields of this glycerol-induced refolding were similar to those observed with ATP. Other polyols such as sucrose, 1,2-propanediol, or 1,3-propanediol also facilitated nucleotide-independent refolding of GS from chaperonin complex. The observed phenomenon cannot be attributed to the viscosity or molecular crowding effects because solutions of dextran or Ficoll with the same viscosity as 4 M glycerol failed to reactivate GroEL-bound GS. Like glycerol, other osmolytes such as betaine and sarcosine or high salt (500 mM NaCl) facilitated spontaneous folding of GS. However, no reactivation of GroEL-bound GS was observed with these additives. The presence of glycerol affected binding of fluorescent probe 1,8-anilinonaphthalene to GroEL, suggesting that glycerol may alter the chaperonin structure. Our data suggest that low-molecular-weight polyols affect both GroEL and bound GS monomers to reduce their binding affinity. This results in an increased partitioning of GS toward active, assembly-competent states.