alpha-crystallin prevents irreversible protein denaturation and acts cooperatively with other heat-shock proteins to renature the stabilized partially denatured protein in an ATP-dependent manner.

alpha-Crystallin, a major lens protein of approximately 800 kDa with subunits of approximately 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced aggregation. Here it is demonstrated that alpha-crystallin can bind to partially denatured enzymes at 42-43 degrees C and prevent their irreversible aggregation, but cannot prevent loss of enzyme activity. However, the alpha-crystallin-bound enzymes regain activity on interaction with other chaperones. The data indicate that the re-activated enzymes are no longer associated with the alpha-crystallin, and ATP is required for re-activation. When inactive luciferase bound to alpha-crystallin was treated with reticulocyte lysate, a rich source of chaperones, up to 60% of the original luciferase activity could be recovered. Somewhat less re-activation was observed when the alpha-crystallin-bound enzyme was treated with heat-shock protein (HSP)70, HSP40, HSP60 and an ATP-generating system. Similar results were also obtained with citrate synthase. The overall results suggest that alpha-crystallin acts to stabilize denaturing proteins so that they can later be re-activated by other chaperones.     

Molecular chaperone function of the Rana catesbeiana small heat shock protein, hsp30

Eukaryotic small heat shock proteins (shps) act as molecular chaperones by binding to denaturing proteins, preventing their heat-induced aggregation and maintaining their solubility until they can be refolded back to their normal state by other chaperones. In this study we report on the functional characterization of a developmentally regulated shsp, hsp30, from the American bullfrog, Rana catesbeiana. An expression vector containing the open reading frame of the hsp30 gene was expressed in Escherichia coli. Purified recombinant hsp30 was recovered as multimeric complexes and was composed of a mixture of alpha-helical and beta-sheet-like structures as determined by circular dichroism analysis. Hsp30 displayed chaperone activity since it inhibited heat-induced aggregation of citrate synthase. Furthermore hsp30 maintained heat-treated luciferase in a folding competent state. For example, heat denatured luciferase when microinjected into Xenopus oocytes did not regain enzyme activity whereas luciferase heat denatured with hsp30 regained 100% enzyme activity. Finally, hsp30 protected the DNA restriction endonuclease, PstI, from heat inactivation. PstI incubated alone at 42 degrees C lost its enzymatic function after 1 h whereas PstI supplemented with hsp30 accurately digested plasmid DNA after 4 h at the elevated temperature. These results clearly indicate a molecular chaperone role for R. catesbeiana hsp30.     

Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase

In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL/GroES-assisted refolding of denatured protein, the yield of correctly folded protein from glycerol- or proline-assisted spontaneous refolding process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance.     

Characterization of the Escherichia coli YedU protein as a molecular chaperone

We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein. The purified protein shows a single band at 31 kDa on SDS-polyacrylamide gels and forms dimers in solution. Like other chaperones, YedU interacts with unfolded and denatured proteins. It promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions. YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity. We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.     

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.     

DsbG, a protein disulfide isomerase with chaperone activity

DsbG, a protein disulfide isomerase present in the periplasm of Escherichia coli, is shown to function as a molecular chaperone. Stoichiometric amounts of DsbG are sufficient to prevent the thermal aggregation of two classical chaperone substrate proteins, citrate synthase and luciferase. DsbG was also shown to interact with refolding intermediates of chemically denatured citrate synthase and prevents their aggregation in vitro. Citrate synthase reactivation experiments in the presence of DsbG suggest that DsbG binds with high affinity to early unstructured protein folding intermediates. DsbG is one of the first periplasmic proteins shown to have general chaperone activity. This ability to chaperone protein folding is likely to increase the effectiveness of DsbG as a protein disulfide isomerase.     

The early-onset torsion dystonia-associated protein,torsinA, displays molecular chaperone activity in vitro

TorsinA is a member of the AAA+ ATPase family of proteins and, notably, is the only known ATPase localized to the ER lumen. It has been suggested to act as a molecular chaperone, while a mutant form associated with early-onset torsion dystonia, a dominantly inherited movement disorder, appears to result in a net loss of function in vivo. Thus far, no studies have examined the chaperone activity of torsinA in vitro. Here we expressed and purified both wild-type (WT) and mutant torsinA fusion proteins in bacteria and examined their ability to function as molecular chaperones by monitoring suppression of luciferase and citrate synthase (CS) aggregation. We also assessed their ability to hold proteins in an intermediate state for refolding. As measured by light scattering and SDS-PAGE, both WT and mutant torsinA effectively, and similarly, suppressed protein aggregation compared to controls. This function was not further enhanced by the presence of ATP. Further, we found that while neither form of torsinA could protect CS from heat-induced inactivation, they were both able to reactivate luciferase when ATP and rabbit reticulocyte lysate were added. This suggests that torsinA holds luciferase in an intermediate state, which can then be refolded in the presence of other chaperones. These data provide conclusive evidence that torsinA acts as a molecular chaperone in vitro and suggests that early-onset torsion dystonia is likely not a consequence of a loss in torsinA chaperone activity but might be an outcome of insufficient torsinA localization at the ER to manage protein folding or trafficking.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.

The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 degrees C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.     

The Hsc66-Hsc20 Chaperone System in Escherichia coli: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions.     

Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery

Small heat shock proteins (hsps) act as molecular chaperones by preventing the thermal aggregation and unfolding of cellular protein; however, the manner by which cells regulate chaperone activity remains unclear. In the present study, we examined the role of phosphorylation on the chaperone function of the Xenopus small hsp30. Both heat stress and sodium arsenite treatment in A6 cells resulted in a rapid activation of p38alpha and MAPKAPK-2. Surprisingly, the association of MAPKAPK-2 with hsp30 and its subsequent phosphorylation were more prevalent during recovery after heat stress. Treatment of A6 cells with SB203580, an inhibitor of the p38 MAP kinase pathway, resulted in a loss of hsp30 phosphorylation. Phosphorylation resulted in the formation of smaller multimeric hsp30 complexes and resulted in a significant loss of secondary structure. Consequently the phosphorylation-induced structural changes severely compromised the ability of hsp30 to prevent the heat-induced aggregation of citrate synthase and luciferase in vitro. We confirmed that the loss of chaperone activity was coincident with an attenuated binding of phosphorylated hsp30 with target proteins. Our data suggest that phosphorylation may be necessary to regulate the post-heat stress molecular chaperone activity of hsp30.     

Biochemical analysis of a cytosolic small heat shock protein, NtHSP18.3, from Nicotiana tabacum

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as 70 degrees . Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.     

Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli

The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31. This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein. In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm.     

S100A1 is a novel molecular chaperone and a member of the Hsp70/Hsp90 multichaperone complex

Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain. In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells. S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway. In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23. The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein. The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock. The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/alpha-crystallin protein

Encysted embryos of the primitive crustacean Artemia franciscana are among the most resistant of all multicellular eukaryotes to environmental stress, in part due to massive amounts of a small heat shock/alpha-crystallin protein (p26) that acts as a molecular chaperone. These embryos also contain very large amounts of the disaccharide trehalose, well known for its ability to protect macromolecules and membranes against damage due to water removal and temperature extremes. Therefore, we looked for potential interactions between trehalose and p26 in the protection of a model substrate, citrate synthase (CS), against heat denaturation and aggregation and in the restoration of activity after heating in vitro. Both trehalose and p26 decreased the aggregation and irreversible inactivation of CS at 43 degrees C. At approximate physiological concentrations (0.4 M), trehalose did not interfere with the ability of p26 to assist in the reactivation of CS after heating, but higher concentrations (0.8 M) were inhibitory. We also showed that CS and p26 interact physically during heating and that trehalose interferes with complex formation and disrupts CS-p26 complexes that form at high temperatures. We suggest from these results that trehalose may act as a "release factor," freeing folding intermediates of CS that p26 can chaperone to the native state. Trehalose and p26 can act synergistically in vitro, during and after thermal stress, suggesting that these interactions also occur in vivo.     

GroE facilitates refolding of citrate synthase by suppressing aggregation.

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.     

Chaperone activity of human small heat shock protein-GST fusion proteins

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.     

Fibrinogen has chaperone-like activity

Partially or completely unfolded polypeptides are highly prone to aggregation due to nonspecific interactions between their exposed hydrophobic surfaces. Extracellular proteins are continuously subjected to stresses conditions, but the existence of extracellular chaperones remains largely unexplored. The results presented here demonstrate that one of the most abundant extracellular proteins, fibrinogen has chaperone-like activity. Fibrinogen can specifically bind to nonnative form of citrate synthase and inhibit its thermal aggregation and inactivation in an ATP-independent manner. Interestingly, fibrinogen maintains thermal-denatured luciferase in a refolding competent state allowing luciferase to be refolded in cooperation with rabbit reticulocyte lysate. Fibrinogen also inhibits fibril formation of yeast prion protein Sup35 (NM). Furthermore, fibrinogen rescues thermal-induced protein aggregation in the plasma of fibrinogen-deficient mice. Our studies demonstrate the chaperone-like activity of fibrinogen, which not only provides new insights into the extracellular chaperone protein system, but also suggests potential diagnostic and therapeutic approaches to fibrinogen-related pathological conditions.     

ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells

DnaK-DnaJ-GrpE and GroEL-GroES are the best-characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat-shock conditions. We show that the absence of either CIpB or HtpG at 42 degrees C leads to increased aggregation of preS2-beta-galactosidase, a fusion protein whose folding depends on DnaK-DnaJ-GrpE, but not GroEL-GroES. However, only the deltaclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL-GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation-prone protein preS2-S'-beta-galactosidase in wild-type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK-DnaJ, but not GroEL-GroES, restore efficient folding in deltaclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK-DnaJ-GrpE team to interact with newly synthesized polypeptides.