Structural and functional diversity of novel and known bacteriophage-encoded chaperonins.

A bioinformatics analysis of the currently predicted GroEL-like proteins encoded by bacteriophage genomes was carried out in comparison with the phage double-ring EL and single-ring OBP chaperonins, previously described by us, as well as with the known chaperonins of group I and group II. A novel GroEL-like protein predicted in the genome of phage AR9 Bacillus subtilis was expressed in E. coli cells, purified and characterised by various physicochemical methods. As shown by native electrophoresis, analytical ultracentrifugation and single-particle electron microscopy analysis, the putative AR9 chaperonin is a single-ring heptamer. Like the EL and OBP chaperonins, the new AR9 chaperonin possesses chaperone activity and does not require co-chaperonin to function. It was shown to prevent aggregation and provide refolding of the denatured substrate protein, endolysin, in an ATP-dependent manner. A comparison of its structural and biochemical properties with those of the EL and OBP chaperonins suggests outstanding diversity in this group of phage chaperonins.

Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay.

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases.

Purification, crystallization, and preliminary X-ray crystallographic analysis of the Group III chaperonin from Carboxydothermus hydrogenoformans.

Chaperonins (CPNs) are megadalton sized ATP-dependent nanomachines that facilitate protein folding through complex cycles of complex allosteric articulation. They consist of two back-to-back stacked multisubunit rings. CPNs are usually classified into Group I and Group II. Here, we report the crystallization of both the AMPPNP (an ATP analogue) and ADP bound forms of a novel CPN, classified as belonging to a third Group, recently discovered in the extreme thermophile Carboxydothermus hydrogenoformans. Crystals of the two forms were grown by the vapor batch crystallization method at 295 K. Crystals of the Ch-CPN/AMPPNP complex diffracted to 3.0 Å resolution and belonged to the space group P422, with unit-cell parameters a = b = 186.166, c = 160.742 Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 60.02%. Crystals of the Ch-CPN/ADP complex diffracted to 4.0 Å resolution and belonged to the space group P4212, with unit-cell parameters a = b = 209.780, c = 169.813Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 70.19%.

Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates.

Crystallization and preliminary X-ray crystallographic analysis of the XoGroEL chaperonin from Xanthomonas oryzae pv. oryzae.

Along with the co-chaperonin GroES, the chaperonin GroEL plays an essential role in enhancing protein folding or refolding and in protecting proteins against misfolding and aggregation in the cellular environment. The XoGroEL gene (XOO_4288) from Xanthomonas oryzae pv. oryzae was cloned and the protein was expressed, purified and crystallized. The purified XoGroEL protein was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to a resolution of 3.4 Å. The crystal belonged to the orthorhombic space group P212121 with 14 monomers in the asymmetric unit, with a corresponding VM of 2.7 Å(3) Da(-1) and a solvent content of 54.5%.

Varied effects of Pyrococcus furiosus prefoldin and P. furiosus chaperonin on the refolding reactions of substrate proteins.

Prefoldin is a molecular chaperone found in the archaeal and eukaryotic cytosol. Prefoldin can stabilize tentatively nascent polypeptide chains or non-native forms of mainly cytoskeletal proteins, which are subsequently delivered to group II chaperonin to accomplish their precise folding. However, the detailed mechanism is not well known, especially with regard to endogenous substrate proteins. Here, we report the effects of Pyrococcus furiosus prefoldin (PfuPFD) on the refolding reactions of Pyrococcus furiosus citrate synthase (PfuCS) and Aequorea enhanced green fluorescence protein (GFPuv) in the presence or absence of Pyrococcus furiosus chaperonin (PfuCPN). We confirmed that both PfuPFD and PfuCPN interacted with PfuCS and GFPuv refolding intermediates. However, the interactions between chaperone and substrate were different for each case, as was the final effect on the refolding reaction. Effects on the refolding reaction varied from passive effects such as ATP-dependent binding and release (PfuCPN towards GFPuv) and binding which leads to folding arrest (PfuPFD towards GFPuv), to active effects such as net increase in thermal stability (PfuCPN towards PfuCS) to an active improvement in refolding yield (PfuPFD towards PfuCS). We postulate that differences in molecular interactions between substrate and chaperone lead to these differences in chaperoning effects.

Chaperonin TRiC assists the refolding of sperm-specific glyceraldehyde-3-phosphate dehydrogenase.

The cytosolic chaperonin TRiC was isolated from ovine testes using ultracentrifugation and heparin-Sepharose chromatography. The molecular mass of the obtained preparation was shown to exceed 900 kDa (by Blue Native PAGE). SDS-PAGE yielded a set of bands in the range of 50-60 kDa. Electron microscopy examination revealed ring-shaped complexes with the outer diameter of 15 nm and the inner diameter of approximately 6 nm. The results suggest that the purified chaperonin is an oligomeric complex composed of two 8-membered rings. The chaperonin TRiC was shown to assist an ATP-dependent refolding of recombinant forms of sperm-specific glyceraldehyde-3-phosphate dehydrogenase, an enzyme that is expressed only in precursor cells of the sperms in the seminiferous tubules of the testes. In contrast, TRiC did not influence the refolding of muscle isoform of glyceraldehyde-3-phosphate dehydrogenase and assisted the refolding of muscle lactate dehydrogenase by an ATP-independent mechanism. The obtained results suggest that TRiC is likely to be involved in the refolding of sperm-specific proteins.

Monoclonal antibodies against a Mycobacterium tuberculosis Ag85B-Hsp16.3 fusion protein.

The secreted Mycobacterium tuberculosis (MTB) proteins, Ag85B and Hsp16.3, have been the focus of intensive research in recent years. These proteins have high sensitivity in bacterium-negative tuberculosis (TB) patients, and are valuable for the rapid diagnosis of bacterium-negative TB. Fusion proteins including multiple antigens such as Ag85B and Hsp16.3 provide improved sensitivity and specificity for serological diagnosis of active TB compared with a single antigen. Many studies have shown that the production of MAbs recognizing a specific repertoire of M. tuberculosis antigens and the tests based on monoclonal antibodies have been found to be valuable in positive detection of TB, particularly for smear-positive pulmonary TB. A number of MAbs are currently used for serodiagnosis of TB. Therefore, an Ag85B-Hsp16.3 fusion protein was expressed and purified using an E. coli system in this study. Three Ag85B-Hsp16.3 fusion protein-specific MAbs were generated by routine murine hybridoma techniques. The titer, specificity, and relative affinity of all three MAbs were determined by ELISA and the serological responses were analyzed. The levels of antigens in a proportion of TB patients were shown to be significantly higher than those in healthy controls. The sensitivity and specificity of the currently available detection systems is likely to be improved by the employment of a combination of these MAbs with others that are already in use.

A single amino acid residue is responsible for species-specific incompatibility between CCT and alpha-actin.

Actin is dependent on the type-II chaperonin CCT (chaperonin containing TCP-1) to reach its native state. In vitro, yeast CCT folds yeast and also mammalian cytoplasmic (beta/gamma) actins but is now found to be incapable of folding mammalian skeletal muscle alpha-actin. Arrest of alpha-actin on yeast CCT at a folding cycle intermediate has been observed by electron microscopy. This discovery explains previous observations in vivo that yeast mutants expressing only the muscle actin gene are non-viable. Mutational analysis identified a single specific alpha-actin residue, Asn-297, that confers this species/isoform folding specificity. The implications of this incompatibility for chaperonin mechanism and actin-CCT co-evolution are discussed.

Chaperone action of a versatile small heat shock protein from Methanococcoides burtonii, a cold adapted archaeon.

The Methanococcoides burtonii small heat shock protein (Mb-sHsp) is an alphaB-crystallin homolog that delivers protein stabilizing and protective functions to model enzymes, presumably reflecting its role as a molecular chaperone in vivo. Although the gene encoding Mb-shsp was cloned from a cold-adapted microorganism, the Mb-sHsp is an efficient protein chaperone at temperatures far above the optimum growth temperature of M. burtonii. We show that Mb-sHsp can prevent aggregation in E. coli cell free extracts at 60 degrees C for 4 h and can stabilize bovine liver glutamate dehydrogenase for 3 h at 50 degrees C. Surface plasmon resonance was used to determine the binding affinity of Mb-sHsp for denatured proteins. Mb-sHsp bound tightly to denatured lysozyme but not to the native form. When Mb-Cpn and Mg(2+)-ATP were added to the reaction, bound lysozyme was released from Mb-sHsp establishing that Mb-Cpn is able to off-load folding intermediates from Mb-sHsp. In addition, Mb-sHsp and Mb-Cpn also function cooperatively to protect an enzyme substrate. Through characterization of these M. burtonii chaperones, we were able to reconstitute a key heat shock regulated protein folding function of this cold adapted organism in vitro.

Bacterial expression and purification of interleukin-2 tyrosine kinase: single step separation of the chaperonin impurity.

Biochemical and biophysical characterization of kinases requires large quantities of purified protein. Here, we report the bacterial expression and purification of active Itk kinase domain (a Tec family kinase) using ArcticExpress cells that co-express the chaperonin system Cpn60/10 from Oleispira antarctica. We describe a simple one step MgCl2/ATP/KCl incubation procedure to remove the co-purifying chaperonin impurity. Chaperonin co-purification is a common problem encountered during protein purification and the simple incubation step described here completely overcomes this problem. The approach targets the chaperonin system rather than the protein of interest and is therefore widely applicable to other protein targets.

A histidine-rich and cysteine-rich metal-binding domain at the C terminus of heat shock protein A from Helicobacter pylori: implication for nickel homeostasis and bismuth susceptibility.

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.

[Prokaryotic expression, purification of HSP65-MUC1 VNTR2 fusion protein and primary research on its tumoricidal effect].

AIM: To express the HSP65-MUC1 VNTR(2) in E.coli and to evaluate its activity of inhibiting tumor growth in vivo. METHODS: HSP65 and MUC1 VNTR(2) were generated by PCR method and sub-cloned to pET28a(+) to construct the recombinant expression vector HSP65-MUC1 VNTR(2)-pET28a(+). E.coli BL21(DE3) bearing the plasmid was induced with IPTG for protein production. Target protein was characterized by Western blot with monoclonal antibody and purified by Q-Sepharose ion-exchange chromatography and gel filtration. The murine cancer cell linejB16 that transfected by human gene MUC1 was utilized to construct the model of carcinoma, and the tumor growth inhibition activities of HSP65-MUC1VNTR(2) was evaluated in mice C57BL/6. RESULTS: The gene HSP65 and MUC1 VNTR(2) confirmed by sequence analysis matched respectively with BCG HSP65 and human gene MUC1 VNTRs in GenBank exactly. The reconstructed vector HSP65-MUC1 VNTR(2)-pET28a could express target protein stably in the soluble fraction of bacterial extract. The purity of HSP65-MUC1 VNTR(2) protein could be above 95% after purification by Q ion-exchange chromatography and gel filtration. The result of Western blot with monoclonal antibody showed positive. The results of prophylactic immunization with HSP65-MUC1 VNTR(2) fusion protein showed that experiment all groups had significantly higher tumor inhibition rates than that of control group. CONCLUSION: In summary, HSP65-MUC1 VNTR(2) fusion protein was solubly expressed in prokaryotic expression system and its tumor growth inhibition activity was evaluated primarily. The result indicated that the fusion protein could inhibit the MUC1 positive tumor growth significantly. It can be used in the future research as the cancer vaccine.

Expression and characterization of the chaperonin molecular machine from the hyperthermophilic archaeon Pyrococcus furiosus.

The chaperonin molecular machine from hyperthermophilic archaeon Pyrococcus furiosus was studied in this paper. The Pyrococcus furiosus chaperonin gene (PfCPN) was amplified by PCR from the Pyrococcus furiosus genomic DNA, and expressed in Escherichia coli BL21-Codonplus(DE)(3)-RIL. The recombinant PfCPN was purified to homogeneity by using ion-exchange and size-exclusion chromatography. It was found that the ATPase activity of the PfCPN was highest at 88 degrees C, and there existed a nested cooperativity of the ATPase activity of the PfCPN. This result suggested that nested allosteric behavior may be common to chaperonin molecular machines from archaea. The half-life (t(1/2)) of the ATPase activity of the PfCPN at 100 degrees C was about 60 min. The PfCPN displayed chaperone activity in preventing lysozyme from thermal inactivation. This chaperone activity was in an ATP-dependent manner.

Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli.

Bacille Calmette-Guerin (BCG)-derived heat shock protein 65 (HSP65) has been demonstrated capable of assisting a fused peptide to generate the peptide-specific cellular immunity. Various HSP65 fusion proteins have been developed as therapeutic cancer vaccines. Purifying a recombinant HSP65 fusion protein with no purification tags for human use is routinely a challenge. Here, we report a scheme for purifying a non-tagged recombinant HSP65-Her2 peptide fusion protein (HSP65-Her2) from Escherichia coli. The HSP65-Her2 is being developed as an immunotherapeutic for the treatment of Her2-positive tumors. After fermentation in a 10-L fermentor, the HSP65-Her2 expressing E. coli were harvested and lysed by sonication. The recombinant HSP65-Her2 was then purified with four successive steps including Butyl-Sepharose FF, DEAE-Sepharose FF, 1% Triton X-114 phase separation and Sephadex G-25. Results showed that HSP65-Her2 was purified up to 97% purity and was able to generate Her2-specific cytotoxic T lymphocytes (CTLs), suggesting that the scheme is efficient for purifying the non-tagged HSP65-Her2 fusion protein with biological activity.

Structure of an Hsp90-Cdc37-Cdk4 complex.

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.

Characterization of the cytoplasmic chaperonin containing TCP-1 from the Antarctic fish Notothenia coriiceps.

The cytoplasmic chaperonin containing TCP-1 (CCT) plays a critically important role in the folding and biogenesis of many cytoskeletal proteins, including tubulin and actin. For marine ectotherms, the chronically cold Southern Ocean (-2 to +2 degrees C) poses energetic challenges to protein folding, both at the level of substrate proteins and with respect to the chaperonin/chaperone folding system. Here we report the partial functional and structural characterization of CCT from an Antarctic notothenioid fish, Notothenia coriiceps. We find that the mechanism of folding by the Antarctic fish CCT differed from that of mammalian CCT: (1) the former complex was able to bind denatured beta-tubulin but (2) when reconstituted with rabbit Cofactor A, failed to release the protein to yield the tubulin/cofactor intermediate. Moreover, the amino acid sequences of the N. coriiceps CCT beta and theta chains contained residue substitutions in the equatorial, apical, and intermediate domains that would be expected to increase the flexibility of the subunits, thus facilitating function of the chaperonin in an energy poor environment. Our work contributes to the growing realization that protein function in cold-adapted organisms reflects a delicate balance between the necessity of structural flexibility for catalytic activity and the concomitant hazard of cold-induced denaturation.

Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit.

The eukaryotic cytosolic chaperonin CCT is an essential ATP-dependent protein folding machine whose action is required for folding the cytoskeletal proteins actin and tubulin, and a small number of other substrates, including members of the WD40-propellor repeat-containing protein family. An efficient purification protocol for CCT from Saccharomyces cerevisiae has been developed. It uses the calmodulin binding peptide as an affinity tag in an internal loop in the apical domain of the CCT3 subunit, which is predicted to be located on the outside of the double-ring assembly. This purified yeast CCT was used for a novel quantitative actin-folding assay with human beta-actin or yeast ACT1p protein folding intermediates, Ac(I), pre-synthesised in an Escherichia coli translation system. The formation of native actin follows approximately a first-order reaction with a rate constant of about 0.03 min(-1). Yeast CCT catalyses the folding of yeast ACT1p and human beta-actin with nearly identical rate constants and yields. The results from this controlled CCT-actin folding assay are consistent with a model where CCT and Ac(I) are in a binding pre-equilibrium with a rate-limiting binding step, followed by a faster ATP-driven processing to native actin. In this pure in vitro system, the human beta-actin mutants, D244S and G150P, show impaired folding behaviour in the manner predicted by our sequence-specific recognition model for CCT-actin interaction.

Affinity purification reveals the association of WD40 protein constitutive photomorphogenic 1 with the hetero-oligomeric TCP-1 chaperonin complex in mammalian cells.

Constitutive photomorphogenic 1 (COP1), a protein composed of a RING finger, a coiled-coil domain and seven WD40 repeats, functions as an E3 ubiquitin ligase that targets key transcription factors for ubiquitination and degradation in both higher plants and mammalian cells. While COP1 is required for light-mediated development in plants, its mammalian counterpart has been implicated in tumorigenesis. We previously showed that COP1 forms high-molecular-weight complexes in mammalian cells. Here we report our attempts in characterizing the components of the mammalian COP1 complexes by affinity purification combined with mass spectral analysis. We find that both transiently and stably expressed COP1 associates with the hetero-oligomeric TCP-1 chaperonin complex (TRiC), heat shock protein 70 (Hsp70) and BAG-family molecular chaperone regulator-2 (BAG2). In addition, stably expressed COP1 binds to major vault protein (MVP) and translocated promoter region (Tpr). The TRiC/Hsp70 complex is known to interact with and assist in the folding of a number of WD40 proteins in Saccharomyces cerevisiae. The association of WD40 protein COP1 with TRiC/Hsp70 in mammalian cells suggests that facilitating the folding of WD40 proteins may be a conserved function for TRiC/Hsp70 from yeast to mammals.

Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.