Natural osmolyte trimethylamine N-oxide corrects assembly defects of mutant branched-chain alpha-ketoacid decarboxylase in maple syrup urine disease

Maple syrup urine disease is caused by deficiency in the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. The clinical phenotype includes often fatal ketoacidosis, neurological derangement, and mental retardation. The type IA mutations Y393N-alpha, Y368C-alpha, and F364C-alpha, which occur in the E1alpha subunit of the decarboxylase (E1) component of the BCKD complex, impede the conversion of an alphabeta heterodimeric intermediate to a native alpha(2)beta(2) heterotetramer in the E1 assembly pathway. In the present study, we show that a natural osmolyte trimethylamine N-oxide (TMAO) at the optimal 1 m concentration restores E1 activity, up to 50% of the wild type, in the mutant E1 carrying the above missense mutations. TMAO promotes the conversion of otherwise trapped mutant heterodimers to active heterotetramers. This slow step does not involve dissociation/reassociation of the mutant heterodimers, which are preformed in the presence of chaperonins GroEL/GroES and Mg-ATP. The TMAO-stimulated mutant E1 activity is remarkably stable upon removal of the osmolyte, when cofactor thiamine pyrophosphate and the transacylase component of the BCKD complex are present. The above in vitro results offer the use of chemical chaperones such as TMAO as an approach to mitigate assembly defects caused by maple syrup urine disease mutations.     

Encapsulation of an 86-kDa assembly intermediate inside the cavities of GroEL and its single-ring variant SR1 by GroES

We described previously that during the assembly of the alpha(2)beta(2) heterotetramer of human mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD), chaperonins GroEL/GroES interact with the kinetically trapped heterodimeric (alphabeta) intermediate to facilitate conversion of the latter to the native BCKD heterotetramer. Here, we show that the 86-kDa heterodimeric intermediate possesses a native-like conformation as judged by its binding to a fluorescent probe 1-anilino-8-naphthalenesulfonate. This large heterodimeric intermediate is accommodated as an entity inside cavities of GroEL and its single-ring variant SR1 and is encapsulated by GroES as indicated by the resistance of the heterodimer to tryptic digestion. The SR1-alphabeta-GroES complex is isolated as a stable single species by gel filtration in the presence of Mg-ATP. In contrast, an unfolded BCKD fusion protein of similar size, which also resides in the GroEL or SR1 cavity, is too large to be capped by GroES. The cis-capping mechanism is consistent with the high level of BCKD activity recovered with the GroEL-alphabeta complex, GroES, and Mg-ATP. The 86-kDa native-like heterodimeric intermediate in the BCKD assembly pathway represents the largest protein substrate known to fit inside the GroEL cis cavity underneath GroES, which significantly exceeds the current size limit of 57 kDa established for unfolded proteins.     

Interactions of GroEL/GroES with a heterodimeric intermediate during alpha 2beta 2 assembly of mitochondrial branched-chain alpha-ketoacid dehydrogenase. cis capping of the native-like 86-kDa intermediate by GroES

We showed previously that the interaction of an alphabeta heterodimeric intermediate with GroEL/GroES is essential for efficient alpha(2)beta(2) assembly of human mitochondrial branched-chain alpha-ketoacid dehydrogenase. In the present study, we further characterized the mode of interaction between the chaperonins and the native-like alphabeta heterodimer. The alphabeta heterodimer, as an intact entity, was found to bind to GroEL at a 1:1 stoichiometry with a K(D) of 1.1 x 10(-)(7) m. The 1:1 molar ratio of the GroEL-alphabeta complex was confirmed by the ability of the complex to bind a stoichiometric amount of denatured lysozyme in the trans cavity. Surprisingly, in the presence of Mg-ADP, GroES was able to cap the GroEL-alphabeta complex in cis, despite the size of 86 kDa of the heterodimer (with a His(6) tag and a linker). Incubation of the GroEL-alphabeta complex with Mg-ATP, but not AMP-PNP, resulted in the release of alpha monomers. In the presence of Mg-ATP, the beta subunit was also released but was unable to assemble with the alpha subunit, and rebound to GroEL. The apparent differential subunit release from GroEL is explained, in part, by the significantly higher binding affinity of the beta subunit (K(D) < 4.15 x 10(-9)m) than the alpha (K(D) = 1.6 x 10(-8)m) for GroEL. Incubation of the GroEL-alphabeta complex with Mg-ATP and GroES resulted in dissociation and discharge of both the alpha and beta subunits from GroEL. The beta subunit upon binding to GroEL underwent further folding in the cis cavity sequestered by GroES. This step rendered the beta subunit competent for reassociation with the soluble alpha subunit to produce a new heterodimer. We propose that this mechanism is responsible for the iterative annealing of the kinetically trapped heterodimeric intermediate, leading to an efficient alpha(2)beta(2) assembly of human branched-chain alpha-ketoacid dehydrogenase.     

GroEL/GroES-dependent reconstitution of alpha2 beta2 tetramers of humanmitochondrial branched chain alpha-ketoacid decarboxylase. Obligatory interaction of chaperonins with an alpha beta dimeric intermediate

The decarboxylase component (E1) of the human mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex (approximately 4-5 x 10(3) kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa alpha subunits and two 37.8-kDa beta subunits. In the present study, His6-tagged E1 alpha2 beta2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 degrees C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 M-1 s-1. Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-alpha and GroEL-beta complexes as combined substrates. However, the beta subunit was markedly more prone to misfolding than the alpha in the absence of GroEL. The alpha subunit was released as soluble monomers from the GroEL-alpha complex alone in the presence of GroES and Mg-ATP. In contrast, the beta subunit discharged from the GroEL-beta complex readily rebound to GroEL when the alpha subunit was absent. Analysis of the assembly state showed that the His6-alpha and beta subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently dimerized to produce the active alpha2 beta2 tetrameter. The purified alpha beta dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-alpha beta ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138 M-1 s-1. Dimers were regenerated from the GroEL-alpha beta complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete. Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 alpha2 beta2 tetramers.     

Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.     

Triggering protein folding within the GroEL-GroES complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.     

Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding

Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.     

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.     

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.     

Insertion mutagenesis of Escherichiacoli GroEL

To gain insights into the in vivo folding and assembly of bacterial chaperonins, groEL was subjected to insertion mutagenesis using transposon ISlacZ/in. Four GroEL-LacZ fusions and the corresponding insertion mutants were obtained after residues 34, 90, 291, and 367. Apical domain insertion mutants GroEL291 and GroEL367 were degraded into monomeric 30- and 40-kDa fragments, respectively. Only the latter was fully soluble, suggesting that proper isomerization of an essentially complete apical domain is required for efficient protomer folding. Truncated variants were inactive as minichaperones as they failed to restore the growth of groEL140 cells at 43 degrees C whether or not GroES was co-expressed. A 31-residue insertion in equatorial helix D led to complete degradation of GroEL90. By contrast, extraneous amino acids were tolerated at equatorial position 34, indicating that this region is highly flexible. Nevertheless, GroEL34 did not fold as efficiently as authentic GroEL and reached only a heptameric conformation.     

Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL

The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.     

GroEL/GroES: structure and function of a two-stroke folding machine

Recent structural and functional studies have greatly advanced our understanding of the mechanism by which chaperonins (Cpn60) mediate protein folding, the final step in the accurate expression of genetic information. Escherichia coli GroEL has a symmetric double-toroid architecture, which binds nonnative polypeptide substrates on the hydrophobic walls of its central cavity. The asymmetric binding of ATP and cochaperonin GroES to GroEL triggers a major conformational change in the cis ring, creating an enlarged chamber into which the bound nonnative polypeptide is released. The structural changes that create the cis assembly also change the lining of the cavity wall from hydrophobic to hydrophilic, conducive to folding into the native state. ATP hydrolysis in the cis ring weakens it and primes the release of products. When ATP and GroES bind to the trans ring, it forms a stronger assembly, which disassembles the cis complex through negative cooperativity between rings. The opposing function of the two rings operates as if the system had two cylinders, one expelling the products of the reaction as the other loads up the reactants. One cycle of the reaction gives the polypeptide about 15 s to fold at the cost of seven ATP molecules. For some proteins, several cycles of GroEL assistance may be needed in order to achieve their native states.     

A mutation in GroEL interferes with protein folding by reducing the rate of discharge of sequestered polypeptides.

GroEL140, a mutant Escherichia coli chaperonin unable to support bacteriophage lambda head assembly, was purified to near homogeneity and compared to wild type GroEL (cpn60). GroEL140 exhibited a 1.5-fold lower ATPase activity relative to the wild type protein. The hydrolysis of ATP by both polypeptides was fully inhibited by an excess of ATP gamma S and partially inhibited by ADP and 5'-adenylylimidodiphosphate, suggesting that adenine nucleotides display different affinities for the ATP binding site of chaperonins. GroEL140 was more sensitive to trypsin digestion compared to wild type GroEL indicating that the mutation destabilized the conformation of the mutant. The proteolytic susceptibility of both chaperonins was similarly enhanced upon addition of ATP, ADP or non-hydrolyzable ATP analogs, providing evidence (i) of a conformational change in the chaperonin structure which is likely to drive the protein discharge process, and (ii) that hydrolysis of ATP is not required to achieve topological modifications. GroEL140 retained its ability to bind non-native ribulose bisphosphate carboxylase/oxygenase (Rbu-P2-carboxylase), but released bound proteins upon addition of ATP and GroES (cpn 10) 6-7-fold less efficiently compared to GroEL. This functional defect was shown to be related to a suboptimal, but not an absence of, interaction with GroES since (i) GroEL140 and GroES were unable to form a complex isolatable by size exclusion chromatography, and (ii) increasing the incubation time or the concentration of GroES enhanced the amount of refolded Rbu-P2-carboxylase discharged from GroEL140-Rbu-P2-carboxylase binary complexes. Pulse-chase experiments involving a double immunoabsorption technique confirmed that Rbu-P2-carboxylase remained associated two times longer with GroEL140 than with GroEL in vivo.     

Coexistence of group I and group II chaperonins in the archaeon Methanosarcina mazei

Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei G?1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.     

A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL.

To facilitate folding and assembly of different proteins, chaperonin GroEL requires the presence of its helper protein GroES. Using a photochemical cross-linking approach, we show that GroES and newly synthesized pre-beta-lactamase (pre-beta lac) contact with each other only within the ternary complex with GroEL. Possibly owing to this contact GroES is able to directly influence the pre-beta lac/GroEL interaction. Furthermore, the cross-linking of pre-beta lac to GroES suggests that the binding of the protein ligands to GroEL occurs near the GroES binding site, known to be in the central hole space of GroEL.     

GroEL-mediated protein folding: making the impossible, possible

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins-constrained by sequence, topology, size, and function-simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

GroEL-Mediated Protein Folding: Making the Impossible,Possible

ABSTRACT

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cell-free translation system (Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. (2005) J. Biol. Chem. 280, 12035-12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroEL-nascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.     

Crystal structure of the native chaperonin complex from Thermus thermophilus revealed unexpected asymmetry at the cis-cavity

The chaperonins GroEL and GroES are essential mediators of protein folding. GroEL binds nonnative protein, ATP, and GroES, generating a ternary complex in which protein folding occurs within the cavity capped by GroES (cis-cavity). We determined the crystal structure of the native GroEL-GroES-ADP homolog from Thermus thermophilus, with substrate proteins in the cis-cavity, at 2.8 A resolution. Twenty-four in vivo substrate proteins within the cis-cavity were identified from the crystals. The structure around the cis-cavity, which encapsulates substrate proteins, shows significant differences from that observed for the substrate-free Escherichia coli GroEL-GroES complex. The apical domain around the cis-cavity of the Thermus GroEL-GroES complex exhibits a large deviation from the 7-fold symmetry. As a result, the GroEL-GroES interface differs considerably from the previously reported E. coli GroEL-GroES complex, including a previously unknown contact between GroEL and GroES.     

Crystallization of the cpn60/cpn10 complex ('holo-chaperonin') from Thermus thermophilus.

A stable complex of the chaperonins, cpn60 and cpn10 (Escherichia coli GroEL and GroES homologues), from the extremely thermophilic bacterium Thermus thermophilus has been isolated and crystallized. The crystals have dimensions up to 30 x 200 x 200 microns. Ultra-thin sections of the crystals estimated by electron microscopy showed a rectangular lattice with unit cell parameters of a = 17 nm, b = 27 nm, gamma = 90 degrees.