GroEL-GroES Complex Research Articles

Effects of Signal Peptide and Chaperone Co-Expression on Heterologous Protein Production in Escherichia coli.

Various host systems have been employed to increase the yield of recombinant proteins. However, some recombinant proteins were successfully produced at high yields but with no functional activities. To achieve both high protein yield and high activities, molecular biological strategies have been continuously developed. This work describes the effect of signal peptide (SP) and co-expression of molecular chaperones on the production of active recombinant protein in Escherichia coli. Extracellular enzymes from Bacillus subtilis, including β-1,4-xylanase, β-1,4-glucanase, and β-mannanase constructed with and without their signal peptides and intracellular enzymes from Pseudomonas stutzeri ST201, including benzoylformate decarboxylase (BFDC), benzaldehyde dehydrogenase (BADH), and d-phenylglycine aminotransferase (d-PhgAT) were cloned and overexpressed in E. coli BL21(DE3). Co-expression of molecular chaperones with all enzymes studied was also investigated. Yields of β-1,4-xylanase (Xyn), β-1,4-glucanase (Cel), and β-mannanase (Man), when constructed without their N-terminal signal peptides, increased 1112.61-, 1.75-, and 1.12-fold, respectively, compared to those of spXyn, spCel, and spMan, when constructed with their signal peptides. For the natural intracellular enzymes, the chaperones, GroEL-GroES complex, increased yields of active BFDC, BADH, and d-PhgAT, up to 1.31-, 4.94- and 37.93-fold, respectively, and also increased yields of Man and Xyn up to 1.53- and 3.46-fold, respectively, while other chaperones including DnaK-DnaJ-GrpE and Trigger factor (Tf) showed variable effects with these enzymes. This study successfully cloned and overexpressed extracellular and intracellular enzymes in E. coli BL21(DE3). When the signal peptide regions of the secretory enzymes were removed, yields of active enzymes were higher than those with intact signal peptides. In addition, a higher yield of active enzymes was obtained, in general, when these enzymes were co-expressed with appropriate chaperones. Therefore, E. coli can produce cytoplasmic and secretory enzymes effectively if only the enzyme coding sequence without its signal peptide is used and appropriate chaperones are co-expressed to assist in correct folding.

Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.

GroEL and the GroEL-GroES Complex.

Chaperonin is categorized as a molecular chaperone and mediates the formation of the native conformation of proteins by first preventing folding during synthesis or membrane translocation and subsequently by mediating the step-wise ATP-dependent release that result in proper folding. In the GroEL-GroES complex, a single heptameric GroEL ring binds one GroES ring in the presence of ATP/ADP, in this vein, the double ring GroEL tetradecamer is present in two distinct types of GroEL-GroES complexes: asymmetric 1:1 "bullet"-shaped GroEL:GroES and symmetric 1:2 "football" (American football)-shaped GroEL:GroES2. There have been debates as to which complex is critical to the productive protein folding mediated by the GroEL-GroES complex, and how GroES coordinates with GroEL in the chaperonin reaction cycle in association with regulation by adenine nucleotides and through the interplay of substrate proteins. A lot of knowledge on chaperonins has been accumulating as if expanding as ripples spread around the GroEL-GroES from Escherichia coli. In this article, an overview is presented on GroEL and the GroEL-GroES complex, with emphasis on their morphological variations, and some potential applications to the fabrication of nanocomposites using GroEL as a nano-block. In parallel, a guideline is presented that supports the recognition that the E. coli and its GroEL-GroES complex do not always receive in standard literature because the biochemical features of chaperonins derived from others special, such as mammals, are not always the same as those confirmed using GroEL-GroES derived from E. coli.

Metal Nanoparticles Dispersion By Chaperonin Complexes

Introduction GroEL is the best characterized chaperonin; it is found in the cytoplasm of Escherichia coli and is essential for cell viability and growth. GroEL is a large cylindrical protein complex comprising two heptameric rings of identical 57-kDa subunits, and these rings are stacked back to back. GroES is a single heptameric ring of identical 10-kDa subunits. GroEL encapsulates the substrates to the cavities, GroEL binds a wide variety of substrate proteins in non-native states, and forms a binary complex, which then binds seven ATP molecules with positive cooperativity and GroES to the same (cis) GroEL ring, to form the cis-ternary complex. The binding of GroES induces the encapsulation of the substrate protein into an enlarged cavity (horizonal width of 5-6nm and vertical width of 6-7nm) inside the cis-ring, which is capped by GroES. In the cis-cavity, non-native protein initiates folding without the risk of aggregation. Following hydrolysis of ATP in the cisring, the products of this cycle, ADP and folded substrate protein, are released with GroES, as another similar folding-active assembly is formed in the opposite trans ring. On the other hand, the hydrolysis-defective mutant GroEL(D52/398A) can bind two GroES in the presence of ATP, producing a symmetric 1:2 GroEL-GroES complex (the “football”-shaped complex) with half-time of ~150 h (~6 days). The property of the GroEL(D52/398A) looks beneficial for dispersing aggregative nanoparticles and hydrophobic compounds. FePt, Pt, CdSe/ZnS and Au were used to encapsulate in GroEL(D52/398A) as metal nanoparicles because these nanoparticles are used to engineering manufactures such as semiconductors, devices and batteries. Experiments Pt (average φ2 nm), FePt (φ4 nm), CdSe/ZnS (φ7nm and 6 nm) and Au (φ10 nm, 4 nm and 2 nm) were used to encapsulate in GroEL(D52/398A) as metal nanoparicles. In the case of nanoparticle encapsulation in one side of GroEL(D52/398A) cavities, 0.1-1.0 µM GroEL(D52/398A), 0.2-2.0 µM GroES (2-fold mol of GroEL), and 1 mM ATP were added to the sonicated nanoparticle solution. For two different nanoparticle encapsulation in both sides of GroEL(D52/398A) cavities, GroEL(D52/398A) which is bound to first nanoparticle was added to the same molar of GroES and 5 mM ADP. The bullet-shaped GroEL(D52/398A) encapsulating first nanoparticle was mixed with the second nanoparticle solution for 1 minute, then, GroES and 1 mM ATP were added to form the football-shaped complex. GroEL(D52/398A) complexes were stained with 1% phosphotungstic acid (pH 4.0) for the observation with the transmission electron microscopy JEM 2000EX or JEM 2100 (JEOL Co., Ltd.). For protein crystalizaion, the sample solution of metal nanoparticle encapsulating GroEL(D52/398A) complexes was added to 1% ammonium molybdate/0.2 % PEG1500 with equal amount volume. Then, the mixture was dropped on TEM grid or flesh cleaved mica and air dried. Fixed protein was observed using with TEM or scanning probe microscopy SPM 9600 (Shimazu Corp.). Analysis was performed using with gel filtration HPLC system equipped with a G3000SWXL (TOSOH) and elution buffer which was consisted of 20 mM HEPES/KOH (pH 7.0), 100 mM KCl, 5 mM MgCl2 and 50 mM Na2SO4 at a flow rate of 0.5 ml/min. The eluate was monitored using a UV detector set at the wavelength 280 nm. Fluorescence intensity was detected at an excitation wavelength of 370 nm and emission at 490 nm for CdSe/ZnS λ490 or 575 nm emission wavelength for CdSe/ZnS λ575 was used. Results and discussion From TEM images, the highest FePt nanoparticles encapsulation percentage of GroEL(D52/398A) was about 95% in this experimental condition. Pt, CdSe/ZnS and Au nanoparticles were encapsulated in GroEL(D52/398A) complexes, too. GroEL(D52/398A) wasn’t seemed to select metal nanoparticle. Two CdSe/ZnS nanoparticles encapsulation of GroEL(D52/398A) of analysis indicated FL emission peaks of both wave length at 490 nm and 575 nm around 12 min of retention time which GroEL(D52/398A) elution time with UV detector. This result showed a possibly that GroEL(D52/398A) could encapsulate two particles in each their cavities. In a presence of protein crystallization reagent, metal nanoparticles encapsulating GroEL(D52/398A) complexes showed tendency to fill up with themselves on TEM grid or mica as an experimental flat plane. These experiments are continued to make monolayer aligns of metal nanoparticle encapsulating GroEL(D52/398A) complexes on flat plated of manufactures. These results from a sequential experiments of GroEL(D52/398A) suggest a potential that GroEL(D52/398A) will support to construct nano manufactures.

3PS029 シャペロニンGroELフットポール型複合体を介したタンパク質フォールディングの直接観察(日本生物物理学会第50回年会(2012年度))

3PS029 シャペロニンGroELフットポール型複合体を介したタンパク質フォールディングの直接観察(日本生物物理学会第50回年会(2012年度))

Single-Molecule Imaging of 1:2 Groel-Groes Complexes in Zero-Mode Waveguides

GroEL is an Escherichia coli chaperonin which is composed of two heptameric rings. GroEL interacts with its cofactor GroES and assists protein folding in an ATP dependent manner. Because of negative cooperativity between two rings of GroEL in the binding of ATP, it has been generally believed that an asymmetrical 1:1 complex is only a functional form for over a decade. Contrary to the belief, we revealed that a symmetrical 1:2 GroEL-GroES complex can be formed in the presence of denatured protein using fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). However, the dynamics of GroEL-GroES interaction including a 1:2 GroEL-GroES complex is still unclear. To clarify this issue, 1:2 GroEL-GroES complexes were observed using zero-mode waveguides (ZMWs) at a single molecule level. ZMWs are nanoholes array fabricated in a thin metal film. They can reduce excitation volume compared to total internal reflection illumination; therefore, they make it possible to observe individual 1:2 GroEL-GroES complexes at sub-μM concentrations that are required to form the complexes. Cy3-GroES and Cy5-GroEL binding to and dissociating from Alexa488-GroES immobilized on the bottoms of ZMWs were visualized. Cy3-GroES and Cy5-GroEL were co-localized in GroES-immobilized ZMWs for ∼ 3 s. The duration time in ZMWs without immobilizing Alexa488-GroES, which reflected non-specific adsorption of GroEL-GroES complex to the bottoms of ZMWs, was ∼ 1 s. These results showed that 1:2 GroEL-GroES complexes were successfully observed at a single-molecule level and their duration time was estimated to be ∼3 s. Furthermore, the same experiment was carried out in the absence of denatured protein. The duration time of Cy3-GroES and Cy5-GroEL in Alexa488-GroES-immobilized ZMWs was ∼1 s. This result indicated that 1:2 GroEL-GroES complexes disappeared in the absence of denatured protein, being consistent with FRET and FCS experiments.

Dissociation Kinetics of the GroEL−gp31 Chaperonin Complex Studied with Förster Resonance Energy Transfer

Propagation of bacteriophage T4 in its host Escherichia coli involves the folding of the major capsid protein gp23, which is facilitated by a hybrid chaperone complex consisting of the bacterial chaperonin GroEL and the phage-encoded co-chaperonin, gp31. It has been well established that the GroEL-gp31 complex is capable of folding gp23 whereas the homologous GroEL-GroES complex cannot perform this function. To assess whether this is a consequence of differences in the interactions of the proteins within the chaperonin complex, we have investigated the dissociation kinetics of GroEL-gp31 and GroEL-GroES complexes using Forster resonance energy transfer. Here we report that the dissociation of gp31 from GroEL is slightly faster than that of GroES from GroEL and is further accelerated by the binding of gp23. In contrast to what had been observed previously, we found that gp23 is able to interact with the GroEL-GroES complex, which might explain how bacteriophage T4 redirects the folding machinery of Escherichia coli during morphogenesis.

Probing Open Conformation of GroEL Rings by Cross-linking Reveals Single and Double Open Ring Structures of GroEL in ADP and ATP

Two heptamer rings of chaperonin GroEL undergo opening-closing conformational transition in the reaction cycle with the aid of GroES and ATP. We introduced Cys into the GroEL subunit at Ala-384 and Ser-509, which are very close between adjacent GroEL subunits in the open heptamer ring but far apart in the closed heptamer ring. The open ring-specific inter-subunit cross-linking between these Cys indicated that the number of rings in open conformation in GroEL was two in ATP (GroEL(OO)), one in ADP (GroEL(O)), and none in the absence of nucleotide. ADP showed an inhibitory effect on ATP-induced generation of GroEL(OO). The isolated GroEL(O) and GroEL(OO), which lost any bound nucleotide, could bind GroES to form a bullet-shaped 1:1 GroEL-GroES complex and a football-shaped 1:2 GroEL-GroES complex, respectively, even without the addition of any nucleotide. Substrate protein was unable to form a stable complex with GroEL(OO) and did not stimulate ATPase activity of GroEL. These results favor a model of the GroEL reaction cycle that includes a football complex as a critical intermediate.

Chaperonin complex with a newly folded protein encapsulated in the folding chamber

A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16-20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23-chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL-GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.

The dynamics of the GroEL‐gp31 chaperonin complex studied with fluorescence spectroscopy

The GroEL‐gp31 chaperonin complex, composed of the E. coli GroEL and the bacteriophage T4 encoded gp31, is essential for the folding of the T4 major capsid protein (gp23). Interestingly the E.coli GroEL‐GroES complex cannot satisfy the folding requirements of gp23. Although the amino acid sequence of gp31 and GroES is only 14% identical, their structure is quite similar. Cryo electron microscopy and image reconstruction of the GroEL‐gp31‐ADP complex revealed that the folding cavity is larger than that of the GroEL‐GroES‐ADP complex (Clare, JMB 2006), which is consistent with the size of the capsid protein (56kDa) being close to the maximum size thought to fit inside the folding cage. The folding of gp23 requires a gp31‐dependant cis‐folding mechanism (Bakkes PNAS 2005) but little is known about the dynamics of the GroEL‐gp31 chaperonin complex. Using stopped‐flow fluorescence spectroscopy, we show that the chaperonin‐assisted refolding of eGFP occurs with the same rate and yield with both GroES and gp31. We also show that the dissociation kinetics of gp31 from GroEL are faster than GroES, and that the binding of the gp23 capsid protein accelerates the ATPase cycle of the GroEL‐gp31 chaperonin complex.

1P036 ADPが対称型1:2GroEL-GroES複合体の形成を阻害する(蛋白質(機能・計測解析の方法論・蛋白質工学),口頭発表,第45回日本生物物理学会年会)

1P036 ADPが対称型1:2GroEL-GroES複合体の形成を阻害する(蛋白質(機能・計測解析の方法論・蛋白質工学),口頭発表,第45回日本生物物理学会年会)

Translocation Boost Protein-Folding Efficiency of Double-Barreled Chaperonins

Incorrect folding of proteins in living cells may lead to malfunctioning of the cell machinery. To prevent such cellular disasters from happening, all cells contain molecular chaperones that assist nonnative proteins in folding into the correct native structure. One of the most studied chaperone complexes is the GroEL-GroES complex. The GroEL part has a “double-barrel” structure, which consists of two cylindrical chambers joined at the bottom in a symmetrical fashion. The hydrophobic rim of one of the GroEL chambers captures nonnative proteins. The GroES part acts as a lid that temporarily closes the filled chamber during the folding process. Several capture-folding-release cycles are required before the nonnative protein reaches its native state. Here we report molecular simulations that suggest that translocation of the nonnative protein through the equatorial plane of the complex boosts the efficiency of the chaperonin action. If the target protein is correctly folded after translocation, it is released. However, if it is still nonnative, it is likely to remain trapped in the second chamber, which then closes to start a reverse translocation process. This shuttling back and forth continues until the protein is correctly folded. Our model provides a natural explanation for the prevalence of double-barreled chaperonins. Moreover, we argue that internal folding is both more efficient and safer than a scenario where partially refolded proteins escape from the complex before being recaptured.

Allosteric signaling of ATP hydrolysis in GroEL–GroES complexes

The double-ring chaperonin GroEL and its lid-like cochaperonin GroES form asymmetric complexes that, in the ATP-bound state, mediate productive folding in a hydrophilic, GroES-encapsulated chamber, the so-called cis cavity. Upon ATP hydrolysis within the cis ring, the asymmetric complex becomes able to accept non-native polypeptides and ATP in the open, trans ring. Here we have examined the structural basis for this allosteric switch in activity by cryo-EM and single-particle image processing. ATP hydrolysis does not change the conformation of the cis ring, but its effects are transmitted through an inter-ring contact and cause domain rotations in the mobile trans ring. These rigid-body movements in the trans ring lead to disruption of its intra-ring contacts, expansion of the entire ring and opening of both the nucleotide pocket and the substrate-binding domains, admitting ATP and new substrate protein.

BeFx Stops the Chaperonin Cycle of GroEL-GroES and Generates a Complex with Double Folding Chambers

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.

High Hydrostatic Pressure Can Probe the Effects of Functionally Related Ligands on the Quaternary Structures of the Chaperonins GroEL and GroES

We investigated the effects of high hydrostatic pressure in the range of 1--3 kilobars on tetradecameric GroEL, heptameric GroES, and the GroEL-GroES complex. Unlike GroEL monomers formed by urea dissociation, which can be reassembled back to the tetradecamer, the pressure-dissociated monomers do not reassemble readily. This indicates an alteration of their native structures, an example of conformational drift. Pressure versus time profiles and kinetics of the dissociation of both GroEL and GroES at fixed pressures were monitored by light scattering. Unlike GroEL, GroES monomers do reassociate readily. Reaction conditions were varied by adding ATP, Mg(2+), ADP, AMP-PNP, and KCl. At any individual pressure, the dissociation process is governed by both thermodynamics and kinetics. This leads to the decrease in the yield of monomers at lower pressures. In the presence of Mg(2+) and KCl, GroEL is stable up to 3 kilobars. The presence of either ATP or ADP but not AMP-PNP leads to GroEL dissociation at lower pressures. Interestingly, the GroEL-GroES complex is very stable in the range of 1--2.5 kilobars. However, the addition of ADP destabilizes the complex, which dissociates completely at 1.5 kilobars. The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer. Such allosteric interactions leading to the alteration of quaternary structure of GroEL in the absence of chemical denaturants are important in understanding the mechanism of chaperonin-assisted protein folding by the GroEL-GroES system.

GroES promotes the T to R transition of the GroEL ring distal to GroES in the GroEL-GroES complex.

Curves of initial rates of ATP hydrolysis by GroEL as a function of ATP concentration, in the presence of fixed concentrations of GroES, were found to deviate from sigmoidal kinetics. Instead of the lag phase typical of sigmoidal curves, a linear phase is observed at low ATP concentrations. Consequently, a good fit of the data to the Hill equation could not be achieved. Such curves could be simulated using a linear combination of Hill equations, thus indicating that more than one allosteric transition is taking place in the ATP concentration range studied. The data were fitted to a fractional saturation equation for ATP binding to GroEL based on a partition function that includes both GroES and ATP-liganded states of GroEL. Using this equation, it was possible to estimate in a reliable manner the value of the allosteric constant, L2', for the transition of the ring distal to GroES in the GroEL-GroES complex from the low (T)- to the high (R)-affinity state for ATP. The value of L2' is found to be 4 x 10(-5) whereas the value of the allosteric constant, L2, for the transition of the second ring of GroEL from the T to R state is 2 x 10(-9) [Yifrach, O., & Horovitz, A. (1995) Biochemistry 34, 5303-5308]. Comparison of these values shows that GroES promotes the T to R transition of the ring distal to GroES in the GroEL-GroES complex. Owing to the relatively low affinity of the R conformation for nonfolded proteins, this transition will lead to release of protein substrates from trans ternary complexes of GroEL, GroES, and protein substrate. The role of this release mechanism may be to assist the folding of relatively large proteins that cannot form cis ternary complexes and/or to facilitate degradation of damaged proteins which cannot fold.

The Chaperonin ATPase Cycle: Mechanism of Allosteric Switching and Movements of Substrate-Binding Domains in GroEL

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by the large chaperonin GroEL, which undergoes major allosteric rearrangements. Interaction between the two back-to-back seven-membered rings of GroEL plays an important role in regulating binding and release of folding substrates and of the small chaperonin GroES. Using cryo-electron microscopy, we have obtained three-dimensional reconstructions to 30 Å resolution for GroEL and GroEL–GroES complexes in the presence of ADP, ATP, and the nonhydrolyzable ATP analog, AMP-PNP. Nucleotide binding to the equatorial domains of GroEL causes large rotations of the apical domains, containing the GroES and substrate protein–binding sites. We propose a mechanism for allosteric switching and describe conformational changes that may be involved in critical steps of folding for substrates encapsulated by GroES.

Putting a lid on protein folding: structure and function of the co-chaperonin, GroES.

The co-chaperonin GroES is an essential partner in protein folding mediated by the chaperonin, GroEL. Two recent crystal structures of GroES provide a structural basis to understand how GroES forms the lid on the folding-active cis ternary complex, and how the GroEL-GroES complex enhances folding.

Biochemical Characterization of Symmetric GroEL-GroES Complexes: EVIDENCE FOR A ROLE IN PROTEIN FOLDING

When chaperonins GroEL and GroES are incubated under functional conditions in the presence of ATP (5 mM) and K+ (150 mM), GroEL-GroES complexes appear in the incubation mixture, that are either asymmetric (1:1 GroEL:GroES oligomer ratio) or symmetric (1:2 GroEL:GroES oligomer ratio). The percentage of symmetric complexes present is directly related to the [ATP]/[ADP] ratio and to the K+ concentration. Kinetic analysis shows that there is a cycle of formation and disappearance of symmetric complexes. A correlation between the presence of symmetric complexes in the incubation mixture and its rhodanese folding activity suggests some active role of these complexes in the protein folding process. Accordingly, under functional conditions, symmetric complexes are found to contain denatured rhodanese. These data suggest that binding of substrate inside the GroEL cavity takes place before the symmetric complex is formed.