Factors affecting procedural pain in children during and immediately after intramuscular botulinum toxin injections for spasticity.

PURPOSE: To evaluate variables that modulate pain during intramuscular botulinum toxin A injections in children. METHODS: As part of a Quality Improvement project, this retrospective analysis compared reported pain during and five minutes post injections with patient and procedural variables using subgroup and regression analyses (N= 593 procedures with 249 unique patients). RESULTS: Mean procedural pain for all procedures (n= 563) was 3.8 ± 3.0. Most children reported no pain (83.8%) or mild pain (12.1%) five minutes after the procedure. Provider, previous patient experience, and dose did not significantly impact pain. Linear regression analysis (R=2 0.64) demonstrated that younger age (p< 0.05), use of vapo-coolant spray or topical anesthetic (p< 0.01), and body region injected (p< 0.01) were significantly associated with increased procedural pain. Logistic regression (R=2 0.14) demonstrated that pain during the procedure (p< 0.001) and older age (p< 0.01) increased the likelihood of pain post-procedure. Utilization of personnel for distraction did not significantly predict pain ratings at either time point. CONCLUSION: Age, topical anesthesia, and injected region impact procedural pain and in nearly 96% of cases, patients report mild or no pain within five minutes. Additional research into these predictors is necessary, but short-lived procedural pain may suggest that frequent use of sedation/anesthesia is unnecessary.

Contributions of different kainate receptor subunits to the properties of recombinant homomeric and heteromeric receptors.

The tetrameric kainate receptors can be assembled from a combination of five different subunit subtypes. While GluK1-3 subunits can form homomeric receptors, GluK4 and GluK5 require a heteromeric partner to assemble, traffic to the membrane surface, and produce a functional channel. Previous studies have shown that incorporation of a GluK4 or GluK5 subunit changes both receptor pharmacology and channel kinetics. We directly compared the functional characteristics of recombinant receptors containing either GluK4 or GluK5 in combination with the GluK1 or GluK2 subunit. In addition, we took advantage of mutations within the agonist binding sites of GluK1, GluK2, or GluK5 to isolate the response of the wild-type partner within the heteromeric receptor. Our results suggest that GluK1 and GluK2 differ primarily in their pharmacological properties, but that GluK4 and GluK5 have distinct functional characteristics. In particular, while binding of agonist to only the GluK5 subunit appears to activate the channel to a non-desensitizing state, binding to GluK4 does produce some desensitization. This suggests that GluK4 and GluK5 differ fundamentally in their contribution to receptor desensitization. In addition, mutation of the agonist binding site of GluK5 results in a heteromeric receptor with a glutamate sensitivity similar to homomeric GluK1 or GluK2 receptors, but which requires higher agonist concentrations to produce desensitization. This suggests that onset of desensitization in heteromeric receptors is determined more by the number of subunits bound to agonist than by the identity of those subunits. The distinct, concentration-dependent properties observed with heteromeric receptors in response to glutamate or kainate are consistent with a model in which either subunit can activate the channel, but in which occupancy of both subunits within a dimer is needed to allow desensitization of GluK2/K5 receptors.

Three dimensional structure of the anthrax toxin translocon-lethal factor complex by cryo-electron microscopy.

We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LF(N) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16 Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LF(N), though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LF(N) extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.

Assembly of anthrax toxin pore: lethal-factor complexes into lipid nanodiscs.

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LFN ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (∼43%). Three-dimensional analysis of negatively stained single particles revealed the LFN -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LFN molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LFN -PA nanodisc structures and use of advanced automated particle selection methods.

Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles.

A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-A resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.

A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment.

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 m) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 degrees C). Glycerol jump experiments with preformed GroEL.PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL.GroES.ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL.GroES.ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.

Complex effects of molecular chaperones on the aggregation and refolding of fibroblast growth factor-1.

Fibroblast growth factor one (FGF-1) exists in a molten globule (MG)-like state under physiological conditions (neutral pH, 37 degrees C). It has been proposed that this form of the protein may be involved in its atypical membrane transport properties. Macromolecular chaperones have been shown to bind to MG states of proteins as well as to be involved in protein membrane transport. We have therefore examined the effect of such proteins on the aggregation and refolding of FGF-1 to evaluate whether they might play a role in FGF-1 transport. The proposed chaperone alpha-crystallin was found to strongly inhibit the aggregation of the MG state of FGF-1. Curiously, two other proteins of similar size and charge (thyroglobulin and a monoclonal IgM immunoglobulin) with no previously reported chaperone properties were also found to have a related effect. In contrast, the chaperone GroEL/ES induced further aggregation of MG-like FGF-1 but had no effect on the native conformation. Both chaperones stimulated refolding to the native state (25 degrees C) but had no detectable effect when FGF-1 was refolded to the MG state (37 degrees C). This suggests that disordered intermediates are present in the folding pathways of the native and MG-like FGF conformations which differ from the MG-like state induced under physiological conditions. FGF-1 does, therefore, interact with molecular chaperones, although this may involve both the MG and the native states of the protein.

Classification and reconstruction of a heterogeneous set of electron microscopic images: a case study of GroEL-substrate complexes.

Image analysis methods were used to separate images of a large macromolecular complex, the chaperonin GroEL, in a preparation in which it is partially liganded to a nonnative protein substrate, glutamine synthetase. The relatively small difference ( approximately 6%) in size between the chaperonin in its free and complexed forms, and the absence of gross changes in overall conformation, made separation of the two types of particles challenging. Different approaches were evaluated and used for alignment and classification of images, both in two common projections and in three dimensions, yielding 2D averages and a 3D reconstruction. The results of 3D analysis describe the conformational changes effected by binding of this particular protein substrate and demonstrate the utility of 2D analysis as an indicator of structural change in this system.

Structural changes in GroEL effected by binding a denatured protein substrate.

In the absence of nucleotides or cofactors, the Escherichia coli chaperonin GroEL binds select proteins in non-native conformations, such as denatured glutamine synthetase (GS) monomers, preventing their aggregation and spontaneous renaturation. The nature of the GroEL-GS complexes thus formed, specifically the effect on the conformation of the GroEL tetradecamer, has been examined by electron microscopy. We find that specimens of GroEL-GS are visibly heterogeneous, due to incomplete loading of GroEL with GS. Images contain particles indistinguishable from GroEL alone, and also those with consistent identifiable differences. Side-views of the modified particles reveal additional protein density at one end of the GroEL-GS complex, and end-views display chirality in the heptameric projection not seen in the unliganded GroEL. The coordinate appearance of these two projection differences suggests that binding of GS, as representative of a class of protein substrates, induces or stabilizes a conformation of GroEL that differs from the unliganded chaperonin. Three-dimensional reconstruction of the GroEL-GS complex reveals the location of the bound protein substrate, as well as complex conformational changes in GroEL itself, both cis and trans with respect to the bound GS. The most apparent structural alterations are inward movements of the apical domains of both GroEL heptamers, protrusion of the substrate protein from the cavity of the cis ring, and a narrowing of the unoccupied opening of the trans ring.

Refolding a glutamine synthetase truncation mutant in vitro: identifying superior conditions using a combination of chaperonins and osmolytes.

A new method that uses a combination of bacterial GroE chaperonins and cellular osmolytes for in vitro protein folding is described. With this method, one can form stable chaperonin-protein folding intermediate complexes to prevent deleterious protein aggregation and, using these complexes, screen a large array of osmolyte solutions to rapidly identify the superior folding conditions. As a test substrate, we used GSDelta468, a truncation mutant of bacterial glutamine synthetase (GS) that cannot be refolded to significant yields in vitro with either chaperones or osmolytes alone. When our chaperonin/osmolyte method was employed to identify and optimize GSDelta468 refolding conditions, 67% of enzyme activity was recovered, comparable with refolding yields of wild type GS. This method can potentially be applied to the refolding of a broad spectrum of proteins.

Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement.

One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

Partitioning of rhodanese onto GroEL. Chaperonin binds a reversibly oxidized form derived from the native protein.

The mammalian mitochondrial enzyme, rhodanese, can form stable complexes with the Escherichia coli chaperonin GroEL if it is either refolded from 8 M urea in the presence of chaperonin or is simply added to the chaperonin as the folded conformer at 37 degreesC. In the presence of GroEL, the kinetic profile of the inactivation of native rhodanese followed a single exponential decay. Initially, the inactivation rates showed a dependence on the chaperonin concentration but reached a constant maximum value as the GroEL concentration increased. Over the same time period, in the absence of GroEL, native rhodanese showed only a small decline in activity. The addition of a non-denaturing concentration of urea accelerated the inactivation and partitioning of rhodanese onto GroEL. These results suggest that the GroEL chaperonin may facilitate protein unfolding indirectly by interacting with intermediates that exist in equilibrium with native rhodanese. The activity of GroEL-bound rhodanese can be completely recovered upon addition of GroES and ATP. The reactivation kinetics and commitment rates for GroEL-rhodanese complexes prepared from either unfolded or native rhodanese were identical. However, when rhodanese was allowed to inactivate spontaneously in the absence of GroEL, no recovery of activity was observed upon addition of GroEL, GroES, and ATP. Interestingly, the partitioning of rhodanese and its subsequent inactivation did not occur when native rhodanese and GroEL were incubated under anaerobic conditions. Thus, our results strongly suggest that the inactive intermediate that partitions onto GroEL is the reversibly oxidized form of rhodanese.

Changing the nature of the initial chaperonin capture complex influences the substrate folding efficiency.

For the chaperonin substrates, rhodanese, malate dehydrogenase (MDH), and glutamine synthetase (GS), the folding efficiencies, and the lifetimes of folding intermediates were measured with either the nucleotide-free GroEL or the activated ATP.GroEL.GroES chaperonin complex. With both nucleotide-free and activated complex, the folding efficiency of rhodanese and MDH remained high over a large range of GroEL to substrate concentration ratios (up to 1:1). In contrast, the folding efficiency of GS began to decline at ratios lower than 8:1. At ratios where the refolding yields were initially the same, only a relatively small increase (1.6-fold) in misfolding kinetics of MDH was observed with either the nucleotide-free or activated chaperonin complex. For rhodanese, no change was detected with either chaperonin complex. In contrast, GS lost its ability to interact with the chaperonin system at an accelerated rate (8-fold increase) when the activated complex instead of the nucleotide-free complex was used to rescue the protein from misfolding. Our data demonstrate that the differences in the refolding yields are related to the intrinsic folding kinetics of the protein substrates. We suggest that the early kinetic events at the substrate level ultimately govern successful chaperonin-substrate interactions and play a crucial role in dictating polypeptide flux through the chaperonin system. Our results also indicate that an accurate assessment of the transient properties of folding intermediates that dictate the initial chaperonin-substrate interactions requires the use of the activated complex as the interacting chaperonin species.

GroE chaperonin-assisted folding and assembly of dodecameric glutamine synthetase.

The folding and assembly of Escherichia coli dodecameric glutamine synthetase is facilitated by the E. coli GroE chaperonins, GroEL and GroES. Since endogenous glutamine synthetase monomers are bound to GroEL immediately after cell lysis and are assembly competent, this strongly suggests that glutamine synthetase is an authentic substrate of the GroE chaperonins. At physiological temperatures, the in vitro reactivation of glutamine synthetase increases from 10 to 70-80% of the original activity when the chaperonin GroEL is included. Although nucleotide binding is sufficient to dissociate assembly competent glutamine synthetase monomers from GroEL, the addition of GroES substantially accelerates the dissociation, assembly, and reactivation. The interactions of glutamine synthetase monomers with the activated chaperonin are transient (t1/2 = 10 sec) and these monomers can be released from GroEL at high concentrations without misfolding or inappropriate aggregation. It has been found that the nucleotide-induced conformational change of GroEL is critical for folding success of glutamine synthetase because the simple displacement of glutamine synthetase monomers from the GroEL chaperonin with another protein substrate inhibits reactivation. During glutamine synthetase refolding, the "high affinity" nucleotide-free GroEL is most efficient in preventing initial folding intermediates from partitioning to off-pathway folding routes. Interestingly, the more physiologically relevant "low affinity" nucleotide-bound ((ATP/ADP) GroEL--GroES) complex is not as efficient at capturing the initial folding intermediates of glutamine synthetase. In contrast to glutamine synthetase, non-authentic "model" substrates such as mammalian mitochondrial rhodanese and mitochondrial malate dehydrogenase show no differences in folding efficiencies with either the "low affinity" or "high affinity" complexes. Besides the nature of the chaperonin complex itself, the mechanism of GroE-assisted folding is determined by the folding environment and, most importantly, by initial interactions of chaperonins with folding intermediates. Glutamine synthetase interacts only transiently with chaperonin complexes, while most of the "model" proteins exhibit relatively long interactions times. It may be indicative of a specific evolutionary selected mechanism of chaperonin-assisted folding (optimizing the folding kinetics), different from that observed with non-authentic chaperonin substrates. Since the kinetics of protein folding depends heavily on the solution environment, studies involving in vivo chaperonin substrates under conditions that closely mimic those found in the cell will be required to define and solve the physiologically relevant kinetic mechanism of chaperonin-assisted folding.

Interactions between the GroE chaperonins and rhodanese. Multiple intermediates and release and rebinding.

Efficient renaturation of urea-denatured rhodanese using the chaperonin GroE system requires GroEL, GroES, and ATP. At high concentrations this renaturation also requires the substrate thiosulfate to have been present during GroEL-rhodanese complex formation. When thiosulfate is present the GroEL-rhodanese complex can be concentrated to greater than 1 mg/ml rhodanese with little effect on the efficiency of renaturation. However, if complex is formed in the absence of thiosulfate, renaturation of rhodanese in the presence of thiosulfate shows a critical concentration of approximately 0.4 mg/ml, above which renaturation yields drop dramatically. This critical concentration appears to be related to an aggregation event in the refolding of rhodanese. The nucleotide free or ADP-bound form of GroEL also binds to rhodanese that has been either already renatured or never denatured. The bound rhodanese has no activity but can be released from GroEL with ATP recovering 90% of control activity. The data presented herein support a release and rebinding mechanism for the GroE-assisted refolding of rhodanese. It also suggests GroEL binds several protein folding intermediates along the entire refolding pathway.

The rates of commitment to renaturation of rhodanese and glutamine synthetase in the presence of the groE chaperonins.

Current models of chaperonin-assisted folding suggest that proteins undergo multiple rounds of binding and release before they are released in a form that is committed to folding to the native state. Using immunoprecipitation techniques, we have determined the rates at which rhodanese and glutamine synthetase (GS) are released from groEL in a form committed to refold to active enzyme. Rhodanese and glutamine synthetase were chosen as substrates because they exhibit different solution requirements for the chaperonin system and they form stable "folding arrested" complexes with groEL. At various times during the groE-dependent renaturations, groEL was rapidly removed from the renaturation mixture by immunoprecipitation and centrifugation (30 s). The conformers that are committed to the native state remained in the supernatant and were assayed after 1 h. At 25 degrees C, the rate profiles indicate the release and commitment to folding of GS to its native state occurs far earlier (t1/2 < 1 min) than for rhodanese (t1/2 = 5 min). In light of previous results, it appears that GS monomers can attain a groE-independent assembly competent conformation after a brief interaction with the chaperonin. In contrast, the renaturation rate for rhodanese with the groE chaperonins mirrored the committed renaturation rates following groEL depletion. This suggests that rhodanese must interact with groEL throughout most of its folding reaction before it acquires a folding competent (groE independent) state. If current models of chaperonin mechanism are correct, rhodanese undergoes more rebinding and release cycles than does GS. Structurally, the degree of cycling and hence the rate of commitment to folding to the active form are probably dictated by the hydrophobic nature, number, and lifetimes of the folding intermediates that interact with the chaperonins.

Refolding and release of tubulins by a functional immobilized groEL column.

Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.

The effect of groES on the groEL-dependent assembly of dodecameric glutamine synthetase in the presence of ATP and ADP.

The yields of active dodecameric glutamine synthetase (GS) are significantly increased when in vitro folding is initiated in the presence of the Escherichia coli groE chaperonins and ATP (37 degrees C). To observe the effects of chaperonins and ATP on GS assembly, the GS assembly intermediates were separated by nondenaturing gel electrophoresis, visualized by Western analysis, and studied as a function of time. The form of GS that was initially released from groEL is monomeric. After the monomers formed dimers, active GS oligomers were assembled by the association of assembly competent dimers with higher order even-numbered oligomers until the dodecamer was formed. When ATP was added to the groEL.GS complex (no groES), a groEL.GS complex remained visible for up to 30 min after the renaturation was initiated. This slow disappearance of the groEL.GS complex is consistent with observed lags in both the GS activity regain profile and the assembly-dependent increase in GS tryptophan fluorescence. When groES was present, the addition of ATP resulted in the disappearance of observable complex at early sample times (< 2 min). Concomitantly, the rates of the regain of GS activity and the GS-dependent increase in tryptophan fluorescence intensity showed substantial accelerations. These results indicate that groES facilitates GS assembly from groEL by inducing the rapid release of GS from groEL, which in turn increases the concentration of assembly competent GS monomers. In addition, groES can initiate renaturation of GS from the groEL.GS arrested complex in the presence of ADP. When chaperonin-dependent GS renaturation was initiated with ATP or ADP (> or = 2 mM), the rates were identical. Since ATP hydrolysis is not absolutely required, the combined binding energies of groES and ATP (or ADP) appear to be sufficient to weaken the binding affinity of groEL for GS subunits and facilitate the release and refolding of assembly competent GS monomers from groEL.

On the assembly of dodecameric glutamine synthetase from stable chaperonin complexes.

For many in vitro protein-folding reactions, the fraction of correctly folded product declines as the initial protein concentration increases due primarily to misfolding and aggregation reactions. Under optimal conditions and in the presence of ATP, chaperonins (groEL and groES) enhanced the renaturation of dodecameric glutamine synthetase (GS) with yields of active enzyme between 75 and 85% of the original activity (Fisher, M.T. (1992) Biochemistry 31, 3955-3963). In spite of this enhancement, a concentration-dependent decline in recoverable activity was observed when increasing concentrations of unfolded GS were rapidly mixed with renaturation buffer containing a 2-fold molar excess (GS subunits:groEL oligomer) of chaperonins. When a stable groEL-GS complex, formed under optimal conditions, was concentrated 4-fold by centrifugal ultrafiltration prior to ATP addition, the amount of total active GS (percent of the original activity) recovered remained at optimal levels and no longer showed a concentration-dependent decline. The GS subunits that are initially bound and then released from groEL by ATP are assembly-competent. It is proposed that the subunits are no longer able to kinetically equilibrate with folding intermediates that misfold or aggregate. If a stable groEL-protein substrate complex can be amassed without loss of activity, this will facilitate studies on molecular aspects of chaperonin release mechanisms and oligomeric protein assembly.