A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL.

Single-point mutants of GroEL were constructed with tryptophan replacing a tyrosine residue in order to examine nucleotide-induced structural transitions spectrofluorometrically. The tyrosine residues at positions 203, 360, 476 and 485 were mutated. Of these, the probe at residue 485 gave the clearest fluorescence signals upon nucleotide binding. The probe at 360 reported similar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated timescales, all of which precede hydrolysis of the nucleotide. All four of these rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is proposed to be a T-to-R transition. The next kinetic phase is a rearrangement to the open state identified by electron cryo-microscopy and this we designate an R to R* transition. Both of these rearrangements can occur when only a single ring of GroEL is loaded with ATP, and the results are consistent with the occupied ring behaving in a concerted, cooperative manner. At higher ATP concentrations both rings can be loaded with the nucleotide and the R to R* transition is accelerated. The resultant GroEL:ATP14 species can then undergo two final rearrangements, RR*-->[RR](+)-->[RR](#). These final slow steps are completely blocked when ADP occupies the second ring, i.e. it does not occur in the GroEL:ATP7:ADP7 or the GroEL:ATP7 species. All equilibrium and kinetic data conform to a minimal model in which the GroEL ring can exist in five distinct states which then give rise to seven types of oligomeric conformer: TT, TR, TR*, RR, RR*, [RR](+) and [RR](#), with concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by inter-ring contacts. This kinetic behaviour is consistent with the GroEL ring passing through distinct functional states in a binding-encapsulation-folding process, with the T-form having high substrate affinity (binding), the R-form being able to bind GroES but retaining substrate affinity (encapsulation), and the R*-form retaining high GroES affinity but allowing the substrate to dissociate into the enclosed cavity (folding). ADP induces only one detectable rearrangement (designated T to T*) which has no properties in common with those elicited by ATP. However, asymmetric ADP binding prevents ATP occupying both rings and, hence, restricts the system to the T*T, T*R and T*R* complexes.

Structure and function in GroEL-mediated protein folding.

Recent structural and biochemical investigations have come together to allow a better understanding of the mechanism of chaperonin (GroEL, Hsp60)-mediated protein folding, the final step in the accurate expression of genetic information. Major, asymmetric conformational changes in the GroEL double toroid accompany binding of ATP and the cochaperonin GroES. When a nonnative polypeptide, bound to one of the GroEL rings, is encapsulated by GroES to form a cis ternary complex, these changes drive the polypeptide into the sequestered cavity and initiate its folding. ATP hydrolysis in the cis ring primes release of the products, and ATP binding in the trans ring then disrupts the cis complex. This process allows the polypeptide to achieve its final native state, if folding was completed, or to recycle to another chaperonin molecule, if the folding process did not result in a form committed to the native state.

Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL.

The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side. We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.

Binding, encapsulation and ejection: substrate dynamics during a chaperonin-assisted folding reaction.

Mitochondrial malate dehydrogenase (mMDH) folds more rapidly in the presence of GroEL, GroES and ATP than it does unassisted. The increase in folding rate as a function of the concentration of GroEL-ES reaches a maximum at a stoichiometry which is approximately equimolar (mMDH subunits:GroEL oligomer) and with an apparent dissociation constant K' for the GroE acceptor state of at least 1 x 10(-8) M. However, even at chaperonin concentrations which are 4000 x K', i.e. at negligible concentrations of free mMDH, the observed folding rate of the substrate remains at its optimum, showing not only that folding occurs in the chaperonin-mMDH complex but also that this rate is uninhibited by any interactions with sites on GroEL. Despite the ability of mMDH to fold on the chaperonin, trapping experiments show that its dwell time on the complex is only 20 seconds. This correlates with both the rate of ATP turnover and the dwell time of GroES on the complex and is only approximately 5% of the time taken for the substrate to commit to the folded state. The results imply that ATP drives the chaperonin complex through a cycle of three functional states: (1) an acceptor complex in which the unfolded substrate is bound tightly; (2) an encapsulation state in which it is sequestered but direct protein-protein contact is lost so that folding can proceed unhindered; and (3) an ejector state which forces dissociation of the substrate whether folded or not.

Domain behavior during the folding of a thermostable phosphoglycerate kinase.

Bacillus stearothermophilus phosphoglycerate kinase (bsPGK) is a monomeric enzyme of 394 residues comprising two globular domains (N and C), covalently linked by an interdomain alpha-helix (residues 170-185). The molecule folds to the native state in three stages. In the first, each domain rapidly and independently collapses to form an intermediate in which the N-domain is stabilized by 5.1 kcal mol-1 and the C-domain by 3.3 kcal mol-1 over their respective unfolded conformations. The N-domain then converts to a folded state at a rate of 1.2 s-1 (delta GI-F = 3.8 kcal mol-1), followed by the C-domain at 0.032 s-1 (delta GI-F = 12.1 kcal mol-1). It is this last step that limits the rate of acquisition of enzyme activity. In the dynamics of unfolding in water, the N-domain converts to the intermediate state at a rate of 8 x 10(-4) s-1, some 10(7) times faster than the C-domain. Consequently, the most populated intermediate in the folding reaction has a native-like N-domain, while that in the unfolding direction has a native-like C-domain. In a conventional sense, therefore, the folding/unfolding kinetics of bsPGK can be described as random order. Consistent with these observations, cutting the molecule in the interdomain helix produces two, independently stable units comprising residues 1-175 and 180-394. A detailed comparison of their folding behavior with that of the whole molecule reveals that true interdomain contacts are relatively weak, contributing approximately 1.4 kcal mol-1 to the stability of the active enzyme. The only interactions which contribute to the stability of rapidly formed intermediates or to transition states along the productive folding pathways are those within domain cores. Contacts formed either between domains or with the interdomain helix are made only in the folded ground state, but do not constitute a separate step in the folding mechanism. Intriguingly, the most pronounced effect of interdomain contacts on the kinetics of folding is inhibitory; the presence of the C-domain appearing to reduce the effective rate of acquisition of native structure within the N-domain.

Release of both native and non-native proteins from a cis-only GroEL ternary complex.

Protein folding by the double-ring chaperonin GroEL is initiated in cis ternary complexes, in which polypeptide is sequestered in the central channel of a GroEL ring, capped by the co-chaperonin GroES. The cis ternary complex is dissociated (half-life of approximately 15 s) by trans-sided ATP hydrolysis, which triggers release of GroES. For the substrate protein rhodanese, only approximately 15% of cis-localized molecules attain their native form before hydrolysis. A major question concerning the GroEL mechanism is whether both native and non-native forms are released from the cis complex. Here we address this question using a 'cis-only' mixed-ring GroEL complex that binds polypeptide and GroES on only one of its two rings. This complex mediates refolding of rhodanese but, as with wild-type GroEL, renaturation is quenched by addition of mutant GroEL 'traps', which bind but do not release polypeptide substrate. This indicates that non-native forms are released from the cis complex. Quenching of refolding by traps was also observed under physiological conditions, both in undiluted Xenopus oocyte extract and in intact oocytes. We conclude that release of non-native forms from GroEL in vivo allows a kinetic partitioning among various chaperones and proteolytic components, which determines both the conformation and lifetime of a protein.

Chaperonins can catalyse the reversal of early aggregation steps when a protein misfolds.

Chaperonins use energy derived from ATP hydrolysis to enhance the efficiency of protein folding by a mechanism which remains a matter of debate. Here, we show that the kinetics of spontaneous and assisted folding of mitochondrial malate dehydrogenase are quantitatively described by a simple physical model. The protein folds from non-native chains by the slow formation of native-like monomers, which then dimerize to form the active enzyme. Misfolding proceeds by two phases of aggregation: the first is slowly reversible, the second is irreversible. Chaperonins accelerate the dissociation of the first-formed, unstable aggregates through a repeated binding-and-release cycle coupled to ATP hydrolysis. By this catalytic action, they supply the productive folding pathway with monomers, and block the irreversible phase of aggregation, thereby maintaining optimal folding yields even when present in sub-stoichiometric quantities. The hydrolytically active chaperonin is required until the substrate protein has completed the slow transition to its native-like, monomeric state. Both the observed rate of folding and the yield are increased by this mechanism without changing real rates in the productive pathway.

The origins and consequences of asymmetry in the chaperonin reaction cycle.

The binding of nucleotides and chaperonin-10 (cpn10) to the Escherichia coli chaperonin-60 (cpn60) and their effect upon the molecular symmetry has been examined both kinetically and at equilibrium. ATP binds tightly and is hydrolysed on only one heptameric ring of the cpn60 tetradecamer at a time, thus inducing asymmetry in the cpn60 oligomer even in the absence of cpn10. In the absence of cpn10 these seven ATP molecules hydrolyse to form a cpn60:ADP7 complex in which ADP is tightly bound (Kd = 2-7 microM); further ADP binding to form a cpn60:ADP14 complex is weak (K1/2 = 2.3 mM). We conclude that symmetrical nucleotide complexes (with 14 ATP or 14 ADPs) are unstable, demonstrating negative co-operativity between the rings. When cpn60 is mixed with cpn10 and ATP the resultant cpn60:ATP7:cpn10 complex is formed rapidly (the rate constant for cpn10 association is > 4 x 10(7) M-1 s-1) and before ATP is hydrolysed (k = 0.12 s-1 per active subunit) to produce an extremely stable cpn60:ADP7:cpn10 complex. This allows ATP association on the unoccupied ring and nucleotide asymmetry in the double toroid is preserved. In "trapping" experiments, where the cpn60:ADP7:cpn10 is challenged with ATP, cpn10 was observed to dissociate at a rate identical to that of steady-state ATP hydrolysis in the presence of cpn10 (k = 0.042 s-1 per active subunit). The spontaneous decay of cpn60:ADP7:cpn10 and any of the major steady-state complexes, under conditions where free nucleotides had been removed, occurred at a rate tenfold lower than ATP hydrolysis. Since the binding of the non-hydrolysable analogue AMP-PNP was unable to induce dissociation of the co-chaperonin it was concluded that a transient state following ATP hydrolysis is necessary for the rapid dissociation of cpn10, which occurs once in every cycle. Trapping experiments using sub-stoichiometric concentrations of cpn10, relative to cpn60, show an unchanged rate of cpn10 exchange upon ATP hydrolysis, indicating that the formation of a symmetric, "football"-shaped complex in which two molecules of the co-chaperonin are bound to cpn60, is not an obligatory intermediate in the exchange process.

Molecular chaperones: physical and mechanistic properties.

Molecular chaperones can be broadly defined as proteins which interact with non-native states of other protein molecules. This activity is important in the folding of newly synthesized polypeptides and the assembly of multisubunit structures; the maintenance of proteins in unfolded states suitable for translocation across membranes; and the stabilization of inactive forms of proteins which are turned on by cellular signals; and the stabilization of proteins unfolded during cellular stress. The major chaperone classes are hsp60 (including TCP1), hsp70 and hsp90. All these proteins prevent the aggregation of unfolded proteins and the strength of interaction with their protein substrates is modified by the binding and hydrolysis of ATP. Hsp70 is a dimeric and ubiquitous protein which binds its substrates in an extended conformation through hydrophobic interactions. It binds to newly synthesized proteins and is required for protein transport. In its ATP-bound state it has a low protein affinity but when the nucleotide is hydrolysed to give the ADP state the affinity is increased. Hsp70 in E. coli (DnaK) is regulated by two co-proteins: DnaJ (of which there are homologues in eukaryotes) stimulates hydrolysis of ATP and GrpE promotes the dissociation of ADP to allow rebinding of ATP. Thus DnaJ promotes the association of substrate proteins and GrpE promotes dissociation. Hsp60 is a large, tetradecameric protein with a central cavity in which non-native protein structures are proposed to bind. It is essential for the folding of a huge spectrum of unrelated proteins and is present in all biological compartments except the ER. As in hsp70, the binding of ATP stimulates release of the substrate and its hydrolysis restores high binding affinity. It functions in conjunction with a co-protein, cpn10, which enhances its ability to eject proteins during the ATPase cycle. The enhancement of folding yields arises either from the prevention of irreversible aggregation or the ability to unfold misfolded structures and allow further attempts to arrive at the native state. Proteins of the hsp90 class are found associated with inactive or unstable substrate proteins within the cell, thus preventing their aggregation and/or permitting rapid activation.

Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy.

Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis. Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4). GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity. Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations. Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown. Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL. Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees. GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate.

Affinity of chaperonin-60 for a protein substrate and its modulation by nucleotides and chaperonin-10.

The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.

The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding.

mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding. The hydrophilic and stable cMDH is bound by cpn60 but released by Mg-ATP alone, while the hydrophobic and unstable mMDH requires both Mg-ATP and cpn10. Citrate equalises the stability of the native state of the two proteins but has no effect on the co-chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor. The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg-ATP. In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin-assisted folding is 3-4-times faster.

The energetics and cooperativity of protein folding: a simple experimental analysis based upon the solvation of internal residues.

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureus nuclease (SAN), was induced with two denaturants, urea and guanidinium chloride (GuHCl). For each protein, structural transitions were monitored by intrinsic fluorescence intensity changes arising from a unique tryptophan residue. In the case of SAN the single, native tryptophan residue was used, whereas for PGK two versions, one with a tryptophan at position 315 and one at 379, were constructed genetically. The resultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturant concentration was varied. We derive the following simple relationship: -RT ln K = delta Gw + n delta Gs,m[D]/Kden. + [D]) where K is the equilibrium constant describing the distribution of folded and unfolded forms at a given denaturant concentration [D], delta Gw is the free energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. delta Gs,m and Kden. are constants derived empirically from the solvation energies of model compounds and represent the behavior of an average internal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For proteins of known structure these values can easily be derived, and for others, average values in guanidinium chloride (delta Gs,m = 0.775 kcal/mol and Kden. = 5.4 M) or urea (delta Gs,m = 1.198 kcal/mol and Kden. = 25.25 M) can be used in the analysis. Results show that the parameters n and delta Gw are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded protein. This simple analytical treatment allows the "cooperativity" of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irrespective of the denaturant concentration needed to induce the transition.

Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: implications for the mechanism of assisted protein folding.

Cpn60 was labeled with pyrene maleimide in order to follow structural rearrangements in the protein triggered by the binding of nucleotides and cpn10. The conjugate binds ATP, AMP-PNP, and ADP(P(i)) with pyrene fluorescence enhancements of 60%, 60%, and 15%, respectively. In each case, binding is cooperative with half-saturation (K1/2) occurring at 10 microM, 290 microM, and 2500 microM and Hill constants (nH) of 4, 3, and 3, respectively. Inclusion of the co-protein, cpn10, tightens the binding of ATP, AMP-PNP, and ADP(P(i)) to give K1/2 values of 6 microM, 100 microM, and < 0.07 microM, respectively, and cooperativity is increased. Titration of the cpn60/ADP (14-mer) complex with cpn10 (7-mer) gives a stoichiometry of 14:7 with respect to subunits, confirming the molecular asymmetry shown by electron microscopy. Transient kinetics demonstrate that ATP initially forms a weak collision complex with cpn60 (Kd = 4 mM) which isomerizes to the strongly binding state at a rate of 180 s-1. We suggest that the slow structural rearrangement driven by ATP binding is the same event which lowers the affinity of the chaperonin for protein substrates; a suggestion reinforced by the loss of AMP-PNP binding affinity in the presence of an unstructured polypeptide. As such, this rearrangement of cpn60 is analogous to a force-generating step in energy transduction. Measurements of ATP hydrolysis (pH 7.5, 25 degrees C) show that it is slow (0.04 s-1) compared both with the structural rearrangement and with the dissociation of products. This defines the steady-state complex as cpn60/ATP, a form of the chaperonin which binds substrate proteins weakly. The rate of hydrolysis of ATP is stimulated 20-fold upon binding unfolded lactate dehydrogenase, and the yield of folded enzyme is increased even in the absence of cpn10. Addition of this co-protein inhibits hydrolysis on only half of the sites in cpn60 and leads to a faster release of folded LDH. A mechanism for the action of chaperonins is proposed which depends upon cpn60 being cycled between states which have, alternately, low and high affinity for unfolded proteins. This cycle is driven by the binding and hydrolysis of ATP.