Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES).
ABSTRACT: In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal 'substrate' for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.
Project description:In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence and absence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). The refolded yields of active malate dehydrogenase are increased almost 3-fold in the presence of groEL, groES, Mg2+/ATP and K+ ions. Chaperonin-assisted refolding of malate dehydrogenase does not have an absolute requirement for K+ ions but Mg2+/ATP is obligatory. When ATP is replaced by other nucleoside triphosphates, or by non-hydrolysable ATP analogues, assisted refolding is prevented. Optimal chaperonin-assisted refolding requires both groEL and groES homo-oligomers in molar excess over malate dehydrogenase. Kinetic analysis shows that the chaperonins do not catalyse the refolding of malate dehydrogenase but increase the flux of unfolded enzyme through the productive refolding pathway without altering and/or accelerating that pathway. Although not acting as refolding catalysts, the chaperonins are able to assist at least six consecutive cycles of malate dehydrogenase refolding.
Project description:We report the characterization of the first chaperonin (Mm-cpn) from a mesophilic archaeon, Methanococcus maripaludis. The single gene was cloned from genomic DNA and expressed in Escherichia coli to produce a recombinant protein of 543 amino acids. In contrast with other known archaeal chaperonins, Mm-cpn is fully functional in all respects under physiological conditions of 37 degrees C. The complex has Mg(2+)-dependent ATPase activity and can prevent the aggregation of citrate synthase. It promotes a high-yield refolding of guanidinium-chloride-denatured rhodanese in a nucleotide-dependent manner. ATP binding is sufficient to effect folding, but ATP hydrolysis is not essential.
Project description:Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.
Project description:Chaperonins assist folding of many cellular proteins, including essential proteins for cell viability. However, it remains unclear how chaperonin-assisted folding is different from spontaneous folding. Chaperonin GroEL/GroES facilitates folding of denatured protein encapsulated in its central cage but the denatured protein often escapes from the cage to the outside during reaction. Here, we show evidence that the in-cage-folding and the escape occur diverging from the same intermediate complex in which polypeptide is tethered loosely to the cage and partly protrudes out of the cage. Furthermore, denatured proteins in the chaperonin cage are kept in more extended conformation than those initially formed in spontaneous folding. We propose that the formation of tethered intermediate of polypeptide is necessary to prevent polypeptide collapse at the expense of polypeptide escape. The tethering of polypeptide would allow freely mobile portions of tethered polypeptide to fold segmentally.
Project description:Under "permissive" conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5-10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering. When refolded in chloride-free buffer, however, dynamic light scattering was eliminated, refolding became concentration-independent, and the rate of refolding became the same as that in GroEL/GroES. The GroEL/GroES chamber thus appears to function passively toward DM-MBP.
Project description:1. Protein-fluorescence studies indicated that phospholipase C from Bacillus cereus is denatured in solutions of guanidinium chloride. The denaturation was not thermodynamically reversible and followed biphasic kinetics. 2. Guanidinium chloride solutions released the structural Zn2+ from the enzyme and rendered all histidine residues chemically reactive. In the presence of free Zn1+ the enzyme was much more resistant to denaturation. Also, the addition for free Zn2+ to the denatured enzyme induced refolding. 3. The Zn2+-free apoenzyme was much more sensitive to guanidinium chloride than was the native enzyme and the denaturation appeared to be thermodynamically reversible. 4. Guanidinium chloride denaturation was associated with a reversible inactivation of the enzyme. Heat-inactivated, coagulated enzyme was substantially re-activated on dissolution in guanidinium chloride solutions followed by dialysis against a Zn2+-containing buffer.
Project description:Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.
Project description:Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60?4 forms a heterooligomeric Cpn60 complex with Cpn60?1 and Cpn60?1-?3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60? subunits cannot complement the function of Cpn60?4. Furthermore, the unique C-terminus of Cpn60?4 is required for the full activity of the unique Cpn60 complex containing Cpn60?4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates.
Project description: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.
Project description:The bacterial chaperonins are highly sophisticated molecular nanomachines, controlled by the hydrolysis of ATP to dynamically trap and remove from the environment unstable protein molecules that are susceptible to denaturation and aggregation. Chaperonins also act to assist in the refolding of these unstable proteins, providing a means by which these proteins may return in active form to the complex environment of the cell. The Escherichia coli GroE chaperonin system is one of the largest protein supramolecular complexes known, whose quaternary structure is required for segregating aggregation-prone proteins. Over the course of more than two decades of research on GroE, it has become accepted that GroE, more specifically the GroEL subunit, is a "high-tolerance" molecular system, capable of accommodating numerous mutations, while retaining its molecular integrity. In some cases, a given site of mutation was revealed to be absolutely required for GroEL function, providing hints regarding the network of signals and triggers that propel this unique system. In other instances, however, a mutation has produced a more delicate response, altering only part of, or in some cases, only a single facet of, the molecular mechanism, and these mutants have often provided invaluable hints on the extent of the complexity underlying chaperonin-assisted protein folding. In this review, we highlight some examples of the latter type of GroEL mutants which compose the unique "mutational repertoire" of GroEL and touch upon the important clues that each mutant provided to the overall effort to elucidate the details of GroE action.