Absence Of GroES Research Articles

Hydrophilic Residues 526KNDAAD531 in the Flexible C-terminal Region of the Chaperonin GroEL Are Critical for Substrate Protein Folding within the Central Cavity

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.

Hydrogen peroxide induces the dissociation of GroEL into monomers that can facilitate the reactivation of oxidatively inactivated rhodanese

Although, several studies have been reported on the effects of oxidants on the structure and function of other molecular chaperones, no reports have been made so far for the chaperonin GroEL. The ability of GroEL to function under oxidative stress was investigated in this report by monitoring the effects of hydrogen peroxide (H 2O 2) on the structure and refolding activity of this protein. Using fluorescence spectroscopy and light scattering, we observed that GroEL showed increases in exposed hydrophobic sites and changes in tertiary and quaternary structure. Differential sedimentation, gel electrophoresis, and circular dichroism showed that H 2O 2 treated GroEL underwent irreversible dissociation into monomers with partial loss of secondary structure. Relative to other proteins, GroEL was found to be highly resistant to oxidative damage. Interestingly, GroEL monomers produced under these conditions can facilitate the reactivation of H 2O 2-inactivated rhodanese but not urea-denatured rhodanese. Recovery of ∼84% active rhodanese was obtained with either native or oxidized GroEL in the absence of GroES or ATP. In comparison, urea-denatured GroEL, BSA and the refolding mixture in the absence of proteins resulted in the recovery of 72, 50, and 49% rhodanese activity, respectively. Previous studies have shown that GroEL monomers can reactivate rhodanese. Here, we show that oxidized monomeric GroEL can reactivate oxidized rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.

Nested MWC model describes hydrolysis of GroEL without assuming negative cooperativity in binding

Folding assistance and ATPase activity of GroEL are based on the existence of different conformations. In order to characterise these conformations, published data on steady state ATPase activity in the absence of GroES were reanalysed simultaneously in terms of the Nested MWC model. This model is a hierarchical extension of the symmetry-model of Monod et al. [J. Mol. Biol. 12 (1965) 88]. An unique set of GroEL specific parameters was obtained. This set was supported by comparison of predictions arising from this set of values with experimental data for hydrolysis of ATP in the presence of ADP and ATPγS, binding of ATPγS and ADP to GroEL in the absence of ATP, and binding of ATP as monitored by fluorescence labelling. Thus, for the first time, multiple data sets for the interaction of nucleotides with GroEL are described quantitatively by an allosteric model. A noteworthy feature of our model is that no negative cooperativity in ATP binding occurs in accordance to experimental observations. Furthermore, the model also includes the existence of a conformation with very high ATPase activity. Such a conformation might be of importance at a certain stage in the folding cycle.

Minimal and optimal mechanisms for GroE-mediated protein folding.

We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i.e., one that has very low spontaneous refolding yield) for which rate data can be acquired. In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate. This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, 11-fold by ADP and GroES, and 465-fold by ATP and GroES. Optimal refolding activity was observed when the dissociation of GroES from the chaperonin complex was dramatically reduced. Although GroEL minichaperones were able to bind denatured mitochondrial malate dehydrogenase, they were ineffective in enhancing the refolding rate. The spectrum of mechanisms for GroE-mediated protein folding depends on the nature of the substrate. The minimal mechanism for permissive substrates (i.e., having significant yields of spontaneous refolding), requires only binding to the apical domain of GroEL. Slow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone. The optimal mechanism, which requires holoGroEL, physiological amounts of GroES, and ATP hydrolysis, is necessary for the chaperonin-mediated folding of nonpermissive substrates at physiologically relevant rates under conditions in which retention of bound GroES prevents the premature release of aggregation-prone folding intermediates from the chaperonin complex. The different mechanisms are described in terms of the structural features of mini- and holo-chaperones.

GroEL/ES chaperonins protect interferon-gamma against physicochemical stress--study of tertiary structure formation by alpha-casein quenching and ELISA.

Interferon-gamma (IFN-gamma) is a structurally labile cytokine that rapidly denatures upon exposure to acid or heat. Here we show that both acid-denatured (pH 2) and thermally inactivated (50 degrees C) porcine IFN-gamma can be rescued with the Escherichia coli GroEL/ES chaperonin system and ATP, and reassembled into bioactive dimers. At 35 degrees C, spontaneous refolding of acid-denatured IFN-gamma was found to be dependent on the presence of guanidinium hydrochloride (0.15-0.25 M) or NaCl (0.1-0.2 M). Under non-permissive reaction conditions for regain of native structure (low-ionic-strength buffer at 35 degrees C), the yield of IFN-gamma refolded with GroEL/ES/ATP increased about 30-fold above the level of spontaneous refolding. In the absence of GroES, GroEL captured IFN-gamma in a folding-competent complex. Under these conditions, both ATP and alpha-casein induced release of IFN-gamma from GroEL but with the released protein tending to partition into sedimentable aggregates. Only in the presence of GroES, did ATP induce complete discharge of IFN-gamma from GroEL, with the released protein refolded into a conformation that is (a) immunoreactive/bio-active, (b) resistant to precipitation and (c) in a dimeric configuration. Chicken egg albumin and 90-kDa heat-shock protein were inactive in the exertion of any protective effect against physicochemical stress. The precise amount of protein refolded to the native state at different times of the folding reaction was determined by alpha-casein quenching and ELISA. The former is based on the conversion by excess alpha-casein of any population of unfolded IFN-gamma into one that escapes antibody recognition by subsequent ELISA. Since the native dimers, however, are not affected by alpha-casein quenching, immunoreactivity is directly proportional to the yield of correctly refolded protein. The validity of this approach was confirmed by measurement of biological activity. GroEL/ES-meditated reactivation amounted to > 80% both by ELISA and antiviral assay.

Complexes between chaperonin GroEL and the capsid protein of bacteriophage HK97.

The 42 kDa capsid protein of bacteriophage HK97 requires the GroEL and GroES chaperonin proteins of its Escherichia coli host to facilitate correct folding, both in vivo and in vitro. In the absence of GroES and ATP, denatured gp5 forms a stable complex with the 14 subunit GroEL molecule. We characterized the electrophoretic and biochemical properties of this complex. In electrophoresis on a native (nondenaturing) gel, the band of the gp5-GroEL complex shifts to a slower migrating position relative to uncomplexed GroEL. The results show that there is only one subunit of gp5 bound to each GroEL 14-mer and that the shift in band position is due primarily to a change in the overall charge of the complex relative to uncomplexed GroEL, and not to a change in size or shape. GroEL forms similar complexes with proteolytic fragments of gp5, with a series of sequence duplication derivatives of gp5, and with other proteins. Electrophoretic examination of these complexes shows that a band shift occurs with proteins larger than 31-33 kDa but not with smaller proteins. For those proteins that cause a band shift upon complex formation, the magnitude of the shift is correlated with the predicted if the charge of the complex were simply the sum of the charge of GroEL and the charge of the substrate protein. We suggest that binding of a substrate protein to GroEL is accompanied by a net binding of solution cations to the complex, but only in the case of proteins above a minimum size of 31-33 kDa. The gp5-GroEL complex is in an association/dissociation equilibrium, with a binding constant measured in the range of 11-17 microM-1.

Electron Microscopy of the GroEL-GroES Filament

Electron microscopy of a filamentous complex of GroEL and GroES has been performed on negatively stained specimens. The filaments have been formed when GroEL and GroES at relatively low molar ratios (e.g., 1:1 to 1:3) are incubated at room temperature in the presence of magnesium and ATP. At higher molar ratio, the symmetrical GroEL-GroES complex predominates. Within the GroEL-GroES filaments, the same structural rearrangements of GroEL were observed as in the "bullet-shaped" or symmetrical GroEL-GroES complexes. In the absence of GroES, GroEL molecules have no tendency to form linear filaments. The sharing of one GroES by two GroEL molecules might indicate a high plasticity of the GroEL-GroES complex. Preliminary Fourier analysis of filaments of increasing length indicates that they possess an inherent helicity. Using the negative staining-carbon film procedure, two-dimensional (2-D) crystal nucleation induced by ammonium molybdate-polyethylene glycol can create undulatory rows of side-on GroEL molecules, which are able extend as a p2 2-D crystal form, readily distinguishable from the filamentous GroEL-GroES aggregates. Crystallographic image processing indicates that the GroEL 2 × 7-mer itself possesses a dyad screw axis, in accord with existing higher resolution X-ray structure data.

Homologous Proteins with Different Affinities for groEL: THE REFOLDING OF THE ASPARTATE AMINOTRANSFERASE ISOZYMES AT VARYING TEMPERATURES

The homologous cytosolic and mitochondrial isozymes of aspartate aminotransferase (c- and mAspAT, respectively) seem to follow very different folding pathways after synthesis in rabbit reticulocyte lysate, suggesting that the nascent proteins interact differently with molecular chaperones (Mattingly, J. R., Jr., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 26320-26327). In an attempt to discern the structural basis for this phenomenon, we have begun to study the effect of temperature on the refolding of the guanidine hydrochloride-denatured, purified proteins and their interaction with the groEL/groES molecular chaperone system from Escherichia coli. In the absence of chaperones, temperature has a critical effect on the refolding of the two isozymes, with mAspAT being more susceptible than cAspAT to diminishing refolding yields at increasing temperatures. No refolding is observed for mAspAT at physiological temperatures. The molecular chaperones groEL and groES can extend the temperature range over which the AspAT isozymes successfully refold; however, cAspAT can still refold at higher temperatures than mAspAT. In the absence of groES and MgATP, the two isozymes interact differently with groEL, groEL arrests the refolding of mAspAT throughout the temperature range of 0-45 degrees C. Adding only MgATP releases very little mAspAT from groEL; both groES and MgATP are required for significant refolding of mAspAT in the presence of groEL. On the other hand, the extent to which groEL inhibits the refolding of cAspAT depends upon the temperature of the refolding reaction, only slowing the reaction at 0 degrees C but arresting it completely at 30 degrees C. MgATP alone is sufficient to effect the release of cAspAT from groEL at any temperature examined; inclusion of groES along with MgATP has no effect on the refolding yield but does increase the refolding rate at temperatures greater than 15 degrees C. These results demonstrate that groEL can have significantly different affinities for proteins with highly homologous final tertiary and quarternary structures and suggest that dissimilarities in the primary sequence of the protein substrates may control the structure of the folding intermediates captured by groEL and/or the composition of the surfaces through which the folding proteins interact with groEL.

Heat shock-induced phosphorylation of GroEL alters its binding and dissociation from unfolded proteins.

During heat shock of Escherichia coli, the expression of the major molecular chaperone, GroEL, increases; in addition, a small fraction of the GroEL becomes phosphorylated (Sherman, M. Yu., and Goldberg, A. L. (1992) Nature 367, 166-1692). This heat shock-induced phosphorylation was found to enhance 50-100-fold the capacity of GroEL to bind to several denatured proteins, including casein and fetuin. The phosphorylated species in the cell extract bound quantitatively to affinity columns containing these ligands, and treatment of the extract with alkaline phosphatase markedly reduced this binding. Like heat shock (42 degrees C), overproduction of GroEL (5-10-fold) from the multicopy plasmid at low temperature (25 degrees C) increased the phosphorylated fraction, which bound strongly to denatured fetuin. Heat shock of these cells further enhanced phosphorylation, and about 15% of the induced level of GroEL could bind tightly to the fetuin column. The predominant form of the GroEL that bound to the denatured protein columns appeared to contain at least one phosphate on each of its subunits, although multiple phosphorylated subunits were also observed. With fetuin and casein as affinity ligands, only the phosphorylated species bound, and this material dissociated quantitatively upon addition of ATP-Mg2+. With CRAG and histone as the ligands, some unphosphorylated GroEL also bound, but this species (unlike the phosphorylated form) was not released by ATP alone; its release required the addition of the cofactor GroES together with ATP. Thus, the phosphorylation of GroEL during heat shock greatly enhances its ability to bind to certain denatured proteins and stimulates its ATP-dependent dissociation in the absence of GroES. Presumably, these alterations in the properties of a fraction of GroEL aid in the refolding or the degradation of specific damaged polypeptides.

The stability of the molecular chaperonin cpn60 is affected by site-directed replacement of cysteine 518.

Cysteine 518 of the molecular chaperonin cpn60 (groEL) has been replaced with serine (C518S) by site-directed mutagenesis. The resulting mutant chaperonin protein is still functional and it can: (a) arrest the spontaneous folding of rhodanese in the absence of GroES and ATP, (b) assist refolding of the enzyme rhodanese in the presence of GroES and ATP/Mg2+, and (c) permit the urea-induced release and refolding of rhodanese from its complex with cpn60. ATP/Mg2+, alone, could discharge active rhodanese from cpn60 complexes formed with either wild type or C518S. In contrast with wild type cpn60, C518S has: (a) reduced stability of its quaternary structure, (b) reduced ability to reassemble tetradecamers after dissociation by urea; (c) reduced ATPase activity; and (d) more easily exposed hydrophobic surfaces. The data suggest that replacement of Cys-518 with Ser in cpn60 destabilizes its oligomeric structure, but there is no significant effect on cpn60 function or the stability of the monomers formed in urea.

Correlation between the stability of the GroEL-protein ligand complex and the release mechanism.

The protein-protein interactions during GroE-mediated protein refolding are of crucial importance for understanding how the assisted refolding of non-native proteins is achieved. Since GroEL seems to be a rather promiscuous polypeptide-binding protein it is not surprising that conditions for efficient dissociation from GroEL are promiscuous as well. To understand assisted protein refolding it is necessary to elucidate the underlying principles of the different partial steps of the functional cycle. Here we show a correlation between the overall stability of the complex between GroEL and ligand protein and the conditions for functional release from the chaperonin. As a model system, differently denatured species of an antibody Fab fragment were used. While weakly bound Fab fragments are functionally released in the absence of GroES, stably associated non-native forms of the same protein are dependent on the presence of the co-chaperonin for optimal GroE-mediated reactivation, suggesting that complex stability determines the release requirement. However, the observed overall stability of the complex between GroEL and substrate protein may be regarded as the net product of constant binding and rebinding of the ligand protein, once associated with GroEL, as shown by competition experiments.

On the role of groES in the chaperonin-assisted folding reaction. Three case studies.

The mechanism by which correctly folded proteins are recovered from stable complexes with groEL is not well understood. Certain target proteins require ATP and groES, while others seemingly dispense with the cochaperonin. Here, we examine the chaperonin-assisted folding of ribulose-1,5-bisphosphate carboxylase, malate dehydrogenase, and citrate synthase, three proteins that are believed to require both chaperonin components for successful reactivation. Surprisingly, in all cases, the need for groES depended on the folding environment. Under "non-permissive" conditions, where unassisted spontaneous folding could not occur, reactivation to the native state required the complete chaperonin system (e.g. groEL, groES, and MgATP). However, under "permissive" conditions where spontaneous folding could occur groES was no longer mandatory. Instead, upon the addition of ATP alone, all three target proteins could be released from groEL, in a form that was capable of reaching the native state. In the permissive setting, groES merely accelerated the rate of the ATP-dependent release process. The results suggest that the incompletely folded protein species that are released from groEL, in the absence of groES, are not necessarily committed to the native state. Similar to the unassisted folding reaction, they still partition between productive and unproductive folding pathways in an environment-dependent manner. It follows that the mechanistic contribution of the co-chaperonin, groES, and its physiological significance in cellular protein folding, could be entirely missed in a permissive in vitro environment.

Hydrolysis of adenosine 5'-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion.

The potassium-ion activation constant (Kact) for the ATPase activity of Escherichia coli chaperonin groEL is inversely dependent upon the ATP concentration over at least 3 orders of magnitude. The ATPase activity shows positively cooperative kinetics with respect to ATP and K+. Both the K0.5 for ATP and cooperativity (as measured by the Hill coefficient) decrease as the K+ concentration increases. Equilibrium binding studies under conditions where hydrolysis does not occur indicate that MgATP binds weakly to groEL in the absence of K+. In the absence of groES, the K(+)-dependent hydrolysis of ATP by groEL continues to completion. In the presence of groES, the time course for the hydrolysis of ATP by groEL becomes more complex. Three distinct kinetic phases can be discerned. Initially, both heptameric toroids turn over once at the same rate that they do in the absence of groES. This leads to the formation of an asymmetric binary complex, groEL14-MgADP7-groES7, in which 7 mol of ADP is trapped in a form that does not readily exchange with free ADP. In the second phase, the remaining seven sites (containing readily exchangeable ADP) turn over, or have the potential to turn over, at the same rate as they do in the absence of groES, so that the overall rate of hydrolysis is maximally 50%. These remaining sites of the asymmetric binary complex do not hydrolyze all of the available ATP. Instead, the second phase of hydrolysis gives way to a third, completely inhibited state, the onset of which is dependent upon the relative affinities of the remaining sites for MgATP and MgADP.(ABSTRACT TRUNCATED AT 250 WORDS)