Allosteric differences dictate GroEL complementation of E. coli.

GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.

The PDB and protein homeostasis: From chaperones to degradation and disaggregase machines.

This review contains a personal account of the role played by the PDB in the development of the field of molecular chaperones and protein homeostasis, from the viewpoint of someone who experienced the concurrent advances in the structural biology, electron microscopy, and chaperone fields. The emphasis is on some key structures, including those of Hsp70, GroEL, Hsp90, and small heat shock proteins, that were determined as the molecular chaperone concept and systems for protein quality control were emerging. These structures were pivotal in demonstrating how seemingly nonspecific chaperones could assist the specific folding pathways of a variety of substrates. Moreover, they have provided mechanistic insights into the ATPase machinery of complexes such as GroEL/GroES that promote unfolding and folding and the disaggregases that extract polypeptides from large aggregates and disassemble amyloid fibers. The PDB has provided a framework for the current success in curating, evaluating, and distributing structural biology data, through both the PDB and the EMDB.

Protein quality control acts on folding intermediates to shape the effects of mutations on organismal fitness.

What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli's gene encoding dihydrofolate reductase (DHFR) and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: overexpression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding, and degradation orchestrated by PQC through the interaction with folding intermediates.

Small Heat Shock Proteins HSP10 and HSP27 in the Left Ventricular Myocardium in Rats with Arterial Hypertension and Insulin-Dependent Diabetes Mellitus.

We studied the expression of small heat shock proteins HSP10 and HSP27 in left ventricular cardiomyocytes in animals with arterial hypertension, insulin-dependent diabetes mellitus, and their combination. The experiment was performed on 38-week-old male Wistar-Kyoto and 38-57-week-old SHR (spontaneously hypertensive) rats. Insulin-dependent diabetes mellitus was modeled by single parenteral injection of streptozotocin (65 mg/kg). Expression of HSP10 and HSP27 in left ventricular cardiomyocytes was evaluated by immunohistochemical assay. It was found that the content of HSP10 in the left ventricular cardiomyocytes decreased in comparison with the control in case of isolated diabetes mellitus and, on the contrary, increased in case of arterial hypertension combined with diabetes mellitus. The intensity of HSP27 expression decreased in case of 38-week arterial hypertension and a combination of arterial hypertension with diabetes mellitus. However, in case of 57-week arterial hypertension we observed an increase in the content of HSP27 in cardiomyocytes.

Activation of mitochondrial unfolded protein response is associated with Her2-overexpression breast cancer.

PURPOSE: Mitochondrial unfolding protein are abundant in breast cancer cells, but the mechanism by which breast cancer cells resist apoptosis is still not fully elucidated. In this study, we explored the role of mitochondrial unfolded protein response (mtUPR)-related proteins in four types of breast cancer tissues. METHODS: Mitochondrial fractions were taken from four breast cancer tissues (luminal A, luminal B, Her2 -overexpression, and TNBC) and the expression of mitochondrial polyubiquitinated proteins was observed by western blot and ELISA. In addition, the expression of hsp10, hsp60, and clpp in mitochondria was observed by western blot in breast cancer tissues and adjacent tissues, and confirmed by ELISA. The expression levels of hsp10 and hsp60 were correlated with clinicopathological parameters in 114 breast cancer patients. RESULTS: We found an increase in the performance of mitochondrial polyubiquitinated proteins in breast cancer tissues of luminal A, luminal B, Her2-overexpression, and TNBC. The mitochondrial hsp10, hsp60, and clpp are abundantly expressed in breast cancer tissues rather than adjacent noncancerous tissues. The expression levels of mitochondrial hsp10 and hsp60 were highest in histological grade 3 breast cancer tissues. Additionally, mitochondria with high hsp60 expression were more present in Her2-positive tumors. CONCLUSIONS: We observed that mtUPR was specifically activated in breast cancer tissues but inactivated in normal mammary tissue. MtUPR had also exhibited a particular increase in Her2-overexpression tumors but not in ER- or PR-positive tumors. Taken together, we suggested that mtUPR may act as a potential candidate for developing novel Her2-overexpression breast cancer therapy.

Chaperonin GroEL accelerates protofibril formation and decorates fibrils of the Het-s prion protein.

We have studied the interaction of the prototypical chaperonin GroEL with the prion domain of the Het-s protein using solution and solid-state NMR, electron and atomic force microscopies, and EPR. While GroEL accelerates Het-s protofibril formation by several orders of magnitude, the rate of appearance of fibrils is reduced. GroEL remains bound to Het-s throughout the aggregation process and densely decorates the fibrils at a regular spacing of ∼200 Å. GroEL binds to the Het-s fibrils via its apical domain located at the top of the large open ring. Thus, apo GroEL and bullet-shaped GroEL/GroES complexes in which only a single ring is capped by GroES interact with the Het-s fibrils; no evidence is seen for any interaction with football-shaped GroEL/GroES complexes in which both rings are capped by GroES. EPR spectroscopy shows that rotational motion of a nitroxide spin label, placed at the N-terminal end of the first ß-strand of Het-s fibrils, is significantly reduced in both Het-s/GroEL aggregates and Het-s fibrils, but virtually completely eliminated in Het-s/GroEL fibrils, suggesting that in the latter, GroEL may come into close proximity to the nitroxide label. Solid-state NMR measurements indicate that GroEL binds to the mobile regions of the Het-s fibril comprising the N-terminal tail and a loop connecting ß-strands 4 and 5, consistent with interactions involving GroEL binding consensus sequences located therein.

The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E. coli.

The secretory production of heterologous proteins in E. coli has revolutionized biotechnology. Efficient periplasmic production of foreign proteins in E. coli often requires a signal peptide to direct proteins to the periplasm. However, the presence of attached signal peptide does not guarantee periplasmic expression of target proteins. Overproduction of auxiliary proteins, such as chaperones can be a useful approach to enhance protein export. In the current study, three chaperone plasmid sets, including GroEL-GroES (GroELS), Dnak-Dnaj-GrpE (DnaKJE), and trigger factor (TF), were coexpressed in E. coli BL21 (DE3) in a pairwise manner with two pET22-b vectors carrying the recombinant hirudin-PA (Hir) gene and different signal sequences alkaline phosphatase (PhoA) and l-asparaginase II (l-ASP). Overexpression of cytoplasmic combinations of molecular chaperones containing GroELS and DnaKJE with PhoAHir increased the secretory production of PhoAHir by 2.6fold (p < 0.05) and 3.5fold (p < 0.01) compared with their controls, respectively. By contrast, secretory production of PhoAHir significantly reduced in the presence of overexpressed TF (p = 0.02). Further, periplasmic expression of l-ASP was significantly increased only in the presence of DnaKJE (p = 0.04). These findings suggest that using molecular chaperones can be helpful for improving periplasmic expression of Hir. However, tagged signal peptides may affect the physicochemical properties and secondary and tertiary structures of mature Hir, which may alter their interactions with chaperones. Hence, using overexpressed chaperones has various effects on secretory production of PhoAHir and l-ASPHir.

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL.

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

ATPase activity of GroEL is dependent on GroES and it is response for environmental stress in Riemerella anatipestifer.

Riemerella anatipestifer (Ra) is a serious gram-negative pathogen of birds and can cause considerable economic losses. The survival mechanisms of R. anatipestifer in the host and environment remain largely unknown. Previous results have demonstrated that GroEL is a molecular chaperone and an important component of the response to various stresses in most bacteria. This study focused on whether GroEL is implicated in this process in R. anatipestifer. The 1629 bp groEL is highly conserved among other gram-negative bacteria (levels of sequence similarity > 60%). A structural analysis and ATPase activity assay revealed that RaGroEL had weak ATPase activity and that the enzyme activity was temperature and ion dependent. GroES partially enhanced the GroEL ATPase activity in the same temperature range. In addition, we studied the mRNA expression of groEL under abiotic stresses caused by heat shock, pH, salt and hydrogen peroxide. These stresses increased the transcription of groEL to varying degrees. In R. anatipestifer, the ATPase activity of GroEL is dependent on GroES and temperature. The expression of groEL was strongly induced by heat, pH, hydrogen peroxide and salt stress. This study is the first to show that GroEL in R. anatipestifer might play a major role in response to environmental stress.

A type D ferulic acid esterase from Streptomyces werraensis affects the volume of wheat dough pastries.

A type D ferulic acid esterase (FAE) was identified in the culture supernatant of Streptomyces werraensis, purified, sequenced, and heterologously produced in E. coli BL21(DE3)Star by co-expressing chaperones groES-groEL (69 U L-1). The unique enzyme with a mass of about 48 kDa showed no similarity to other FAEs, and only moderate homology (78.5%) to a Streptomycete ß-xylosidase. The purified reSwFAED exhibited a temperature optimum of 40 °C, a pH optimum in the range from pH seven to eight and a clear preference for bulky natural substrates, such as 5-O-trans-feruloyl-L-arabinofuranose (FA) and ß-D-xylopyranosyl-(1→2)-5-O-trans-feruloyl-L-arabinofuranose (FAX), compared to the synthetic standard substrate methyl ferulate. Treatment of wheat dough with as little as 0.03 U or 0.3 U kg-1 reSwFAED activity resulted in a significant increase of the bun volume (8.0 or 9.7%, resp.) after baking when combined with polysaccharide-degrading enzymes from Aspergillus. For the first time, the long-standing, but rarely proven positive effect of a FAE in baking was confirmed.

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs from Galeruca daurica (Coleoptera: Chrysomelidae) and their expression analysis.

Galeruca daurica (Joannis) is a new outbreak pest in the Inner Mongolia grasslands in northern China. Heat shock protein 10 and 60 (Hsp10 and Hsp60) genes of G. daurica, designated as GdHsp10 and GdHsp60, were cloned by rapid amplification of cDNA ends techniques. Sequence analysis showed that GdHsp10 and GdHsp60 encoded polypeptides of 104 and 573 amino acids, respectively. Sequence alignment and phylogenetic analysis clearly revealed that the amino acids of GdHsp10 and GdHsp60 had high homology and were clustered with other Hsp10 and Hsp60 genes in insects which are highly relative with G. daurica based on morphologic taxonomy. The mRNA expression analysis by real-time PCR revealed that GdHsp10 and GdHsp60 were expressed at all development stages and in all tissues examined, but expressed highest in eggs and in adults' abdomen; both heat and cold stresses could induce mRNA expression of GdHsp10 and GdHsp60 in the 2nd instar larvae; the two Hsp genes were expressed from high to low with the extension of treatment time in G. daurica eggs exposed to freezing point. Overall, our study provides useful information to understand temperature stress responses of Hsp60 and Hsp10 in G. daurica, and provides a basis to further study functions of Hsp60/Hsp10 relative to thermotolerance and cold hardiness mechanism.

Biochemical and Genetic Analysis of the Chlamydia GroEL Chaperonins.

Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigm Escherichia coli chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of groEL and groES homologs, groEL paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. Chlamydia spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three groEL genes, Chlamydia groEL (ChgroEL), ChgroEL2, and ChgroEL3 We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel Chlamydia-specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the in vivo essentiality of the three ChgroEL genes through targeted mutagenesis in Chlamydia trachomatis ChGroEL hydrolyzed ATP at a rate 25% of that of EcGroEL and bound with high affinity to ChGroES, and the ChGroEL-ChGroES complex could refold malate dehydrogenase (MDH). The chlamydial ChGroEL was selective for its cognate cochaperonin, ChGroES, while EcGroEL could function with both EcGroES and ChGroES. A P35T ChGroES mutant (ChGroESP35T) reduced ChGroEL-ChGroES interactions and MDH folding activities but was tolerated by EcGroEL. Both ChGroEL-ChGroES and EcGroEL-ChGroESP35T could complement an EcGroEL-EcGroES mutant. Finally, we successfully inactivated both paralogs but not ChgroEL, leading to minor growth defects in cell culture that were not exacerbated by heat stress. Collectively, our results support novel functions for the paralogs and solidify ChGroEL as a bona fide chaperonin that is biochemically distinct from EcGroEL.IMPORTANCEChlamydia is an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL and chaperonin paralog ChGroEL2 have been associated with survival under stress conditions, and ChGroEL is linked with immunopathology elicited by chlamydial infections. However, their exact roles in bacterial survival and disease remain unclear. Our results further substantiate the hypotheses that ChGroEL is the primary chlamydial chaperonin and that the paralogs play specialized roles during infection. Furthermore, ChGroEL and the mitochondrial GroEL only functioned with their cochaperonin, in contrast to the promiscuous nature of GroEL from E. coli and Helicobacter pylori, which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle.

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.

Leishmania donovani chaperonin 10 regulates parasite internalization and intracellular survival in human macrophages.

Protozoa of the genus Leishmania infect macrophages in their mammalian hosts causing a spectrum of diseases known as the leishmaniases. The search for leishmania effectors that support macrophage infection is a focus of significant interest. One such candidate is leishmania chaperonin 10 (CPN10) which is secreted in exosomes and may have immunosuppressive properties. Here, we report for the first time that leishmania CPN10 localizes to the cytosol of infected macrophages. Next, we generated two genetically modified strains of Leishmania donovani (Ld): one strain overexpressing CPN10 (CPN10+++) and the second, a CPN10 single allele knockdown (CPN10+/-), as the null mutant was lethal. When compared with the wild-type (WT) parental strain, CPN10+/- Ld showed higher infection rates and parasite loads in human macrophages after 24 h of infection. Conversely, CPN10+++ Ld was associated with lower initial infection rates. This unexpected apparent gain-of-function for the knockdown could have been explained either by enhanced parasite internalization or by enhanced intracellular survival. Paradoxically, we found that CPN10+/- leishmania were more readily internalized than WT Ld, but also displayed significantly impaired intracellular survival. This suggests that leishmania CPN10 negatively regulates the rate of parasite uptake by macrophages while being required for intracellular survival. Finally, quantitative proteomics identified an array of leishmania proteins whose expression was positively regulated by CPN10. In contrast, many macrophage proteins involved in innate immunity were negatively regulated by CPN10. Taken together, these findings identify leishmania CPN10 as a novel effector with broad based effects on macrophage cell regulation and parasite survival.

The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4(-/-) mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Heterologous Expression of Chaperones from Hyperthermophilic Archaea Inhibits Aminoglycoside-Induced Protein Misfolding in Escherichia coli.

Aminoglycoside antibiotics affect protein translation fidelity and lead to protein aggregation and an increase in intracellular oxidative stress level as well. The overexpression of the chaperonin GroEL/GroES system promotes short-term tolerance to aminoglycosides in Escherichia coli. Here, we demonstrated that the coexpression of prefoldin or Hsp60 originating from the hyperthermophilic archaeon Pyrococcus furiosus in E. coli cells can rescue cell growth and inhibit protein aggregation induced by streptomycin exposure. The results of our study show that hyperthermophilic chaperones endow E. coli with a higher tolerance to streptomycin than the GroEL/GroES system, and that they exert better effects on the reduction of intracellular protein misfolding, indicating that these chaperones have unique features and functions.

Prolonged fasting identifies heat shock protein 10 as a Sirtuin 3 substrate: elucidating a new mechanism linking mitochondrial protein acetylation to fatty acid oxidation enzyme folding and function.

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function.

Enhanced expression of soluble human papillomavirus L1 through coexpression of molecular chaperonin in Escherichia coli.

The major recombinant capsid protein L1 of human papillomavirus (HPV) is widely used to produce HPV prophylactic vaccines. However, the quality of soluble and active expression of L1 in Escherichia coli was below the required amount. Coexpression with the chaperonin GroEL/ES enhanced L1 expression. Overexpressing GroEL/ES increased the soluble expression level of glutathione S-transferase-fused L1 (GST-L1) by approximately ∼3 fold. The yield of HPV type 16 L1 pentamer (L1-p) was ∼2 fold higher than that in a single expression system after purification through size-exclusion chromatograph. The expression and purification conditions were then optimized. The yield of L1-p was enhanced by ∼5 fold, and those of HPV types 18 and 58 L1-p increased by ∼3 and ∼2 folds, respectively, compared with that in the single expression system. Coexpressing the mono-site mutant HPV16 L1 L469A with GroEL/ES increased L1-p yield by ∼7 fold compared with strains expressing the wild-type L1 gene. L1-p was then characterized using circular dichroism spectra, UV-vis cloud point, dynamic light scattering and transmission electron microscope analyses. Results indicated that the conformation and biological characteristics of L1-p were identical to that of native L1. Hence, overexpressing chaperonin in E. coli can increase the expression level of GST-L1 and L1-p production after purification. This finding may contribute to the development of a platform for prophylactic HPV vaccines.

Molecular cloning, characterization and expression analysis of a heat shock protein 10 (Hsp10) from Pennisetum glaucum (L.), a C4 cereal plant from the semi-arid tropics.

Heat shock proteins (Hsp10) belong to the ubiquitous family of heat-shock molecular chaperones found in the organelles of both prokaryotes and eukaryotes. Chaperonins assist the folding of nascent and stress-destabilized proteins. A cDNA clone encoding a 10 kDa Hsp was isolated from pearl millet, Pennisetum glaucum (L.) by screening a heat stress cDNA library. The fulllength PgHsp10 cDNA consisted of 297 bp open reading frame (ORF) encoding a 98 amino acid polypeptide with a predicted molecular mass of 10.61 kDa and an estimated isoelectric point (pI) of 7.95. PgHsp10 shares 70-98 % sequence identity with other plant homologs. Phylogenetic analysis revealed that PgHsp10 is evolutionarily close to the maize Hsp10 homolog. The predicted 3D model confirmed a conserved eight-stranded ß-barrel with active site between the ß-barrel comprising of eight-strands, with conserved domain VLLPEYGG sandwiched between two ß-sheets. The gene consisted of 3 exons and 2 introns, while the position and phasing of these introns were conserved similar to other plant Hsp10 family genes. In silico analysis of the promoter region of PgHsp10 presented several distinct set of cis-elements and transcription factor binding sites. Quantitative RT-PCR analysis showed that PgHsp10 gene was differentially expressed in response to abiotic stresses with the highest level of expression under heat stress conditions. Results of this study provide useful information regarding the role of chaperonins in stress regulation and generated leads for further elucidation of their function in plant stress tolerance.

Substrate protein switches GroE chaperonins from asymmetric to symmetric cycling by catalyzing nucleotide exchange.

The complex kinetics of Pi and ADP release by the chaperonin GroEL/GroES is influenced by the presence of unfolded substrate protein (SP). Without SP, the kinetics of Pi release are described by four phases: a "lag," a "burst" of ATP hydrolysis by the nascent cis ring, a "delay" caused by ADP release from the nascent trans ring, and steady-state ATP hydrolysis. The release of Pi precedes the release of ADP. The rate-determining step of the asymmetric cycle is the release of ADP from the trans ring of the GroEL-GroES1 "bullet" complex that is, consequently, the predominant species. In the asymmetric cycle, the two rings of GroEL function alternately, 180° out of phase. In the presence of SP, a change in the kinetic mechanism occurs. With SP present, the kinetics of ADP release are also described by four phases: a lag, a "surge" of ADP release attributable to SP-induced ADP/ATP exchange, and a "pause" during which symmetrical "football" particles are formed, followed by steady-state ATP hydrolysis. SP catalyzes ADP/ATP exchange on the trans ring. Now ADP release precedes the release of Pi, and the rate-determining step of the symmetric cycle becomes the hydrolysis of ATP by the symmetric GroEL-GroES2 football complex that is, consequently, the predominant species. A FRET-based analysis confirms that asymmetric GroEL-GroES1 bullets predominate in the absence of SP, whereas symmetric GroEL-GroES2 footballs predominate in the presence of SP. This evidence suggests that symmetrical football particles are the folding functional form of the chaperonin machine in vivo.