Related Articles

Early Closure of a Long Loop in the Refolding of Adenylate Kinase: A Possible Key Role of Non-Local Interactions in the Initial Folding Steps.

J Mol Biol. 2008 Nov 5;

Authors: Orevi T, Ben Ishai E, Pirchi M, Jacob MH, Amir D, Haas E

Most globular protein chains, when transferred from high to low denaturant concentrations, collapse instantly before they refold to their native state. The initial compaction of the protein molecule is assumed to have a key effect on the folding pathway, but it is not known whether the earliest structures formed during or instantly after collapse are defined by local or by non-local interactions-that is, by secondary structural elements or by loop closure of long segments of the protein chain. Stable closure of one or several long loops can reduce the chain entropy at a very early stage and can prevent the protein from following non-productive pathways whose number grows exponentially with the length of the protein chain. In Escherichia coli adenylate kinase (AK), about seven long loops define the topology of the native structure. We selected four loop-forming sections of the chain and probed the time course of loop formation during refolding of AK. We labeled the termini of the loop segments with tryptophan and cysteine-5-amidosalicylic acid. This fluorescence resonance excitation energy transfer donor-acceptor pair is suitable for detecting very short distances and thus is able to distinguish between random and specific compactions. Refolding of AK was initiated by stopped-flow mixing, followed simultaneously by donor and acceptor fluorescence, and analyzed in terms of energy transfer efficiency and distance. In the collapsed state of AK, observed after the 5-ms dead time of the instrument, one of the selected segments shows a native-like separation of its termini; it forms a loop already in the collapsed state. A second segment that includes the first but is longer by 15 residues shows an almost native-like separation of its termini. In contrast, a segment that is shorter but part of the second segment shows a distance separation of its termini as high as a segment that spans almost the whole protein chain. We conclude that a specific network of non-local interactions, the closure of one or several loops, can play an important role in determining the protein folding pathway at its early phases.

PMID: 19013178 [PubMed - as supplied by publisher]

Related Articles

Hsp104 and ClpB: protein disaggregating machines.

Trends Biochem Sci. 2008 Nov 11;

Authors: Doyle SM, Wickner S

Heat-shock protein 104 (Hsp104) and caseinolytic peptidase B (ClpB), members of the AAA+ superfamily, are molecular machines involved in disaggregating insoluble protein aggregates, a process not long ago thought to be impossible. During extreme stress they are essential for cell survival. In addition, Hsp104 regulates prion assembly and disassembly. For most of their protein remodeling activities Hsp104 and ClpB work in collaboration with the Hsp70 or DnaK chaperone systems. Together, the two chaperones catalyze protein disaggregation and reactivation by a mechanism probably involving the extraction of polypeptides from aggregates by forced unfolding and translocation through the Hsp104/ClpB central cavity. The polypeptides are then released back into the cellular milieu for spontaneous or chaperone-mediated refolding.

PMID: 19008106 [PubMed - as supplied by publisher]

Chaperone-assisted column refolding of gloshedobin with the use of refolding cocktail.

J Chromatogr A. 2008 Oct 25;

Authors: Nian R, Tan L, Yoo IK, Choe WS

Gloshedobin, a recently isolated thrombin-like enzyme from the snake venom of Gloydius shedaoensis, is expressed mainly in the form of inclusion bodies (IBs) in Escherichia coli due to its cysteine-rich nature. Following extraction and solubilization of the IBs, one-step immobilized metal affinity chromatography purification produces highly purified (>99%) denatured-solubilized gloshedobin ready to enter the subsequent refolding process. However, the traditional dilution or column refolding strategy, based on gradual denaturant removal, is found to be inefficient for the recovery of protein activity. In this study, a new refolding strategy harnessing the ClpB and DnaK/DnaJ/GrpE bichaperone system is demonstrated to be superior to the conventional refolding methods in either batch dilution or column refolding mode. It is noted that the efficacy of bichaperone-mediated column refolding strategy is further highlighted especially when refolding reaction is attempted at a higher protein concentration with the recirculation of the refolding cocktail containing the bichaperone system. This is evidenced by an uncompromised refolding efficiency (ca. 21.4%) achieved at 2000mug/mL of initial protein concentration, which is comparable to the refolding efficiency (ca. 22.5%) obtained at 20 times lower protein concentration (i.e. 100mug/mL) in the conventional batch dilution refolding technique. The demonstrated chaperone-assisted column refolding strategy thus provides an effective tool for refolding-recalcitrant proteins whose reactivation is otherwise difficult to achieve.

PMID: 19004452 [PubMed - as supplied by publisher]

Inducible Heat Shock Protein70 Prevents Multifocal Flat Dysplastic Lesions and Invasive Tumors in an Inflammatory Model of Colon Cancer.

Carcinogenesis. 2008 Nov 12;

Authors: Tao Y, Hart J, Lichtenstein L, Joseph L, Ciancio M, Hu S, Chang EB, Bissonnette M

Background: Heat shock protein70 (Hsp70)(1) regulates protein biosynthesis and refolding of denatured proteins. Since Hsp70 participates in recovery from stress injury, we examined the effect of Hsp70 genetic deletion in the azoxymethane/dextran sulfate sodium (AOM/DSS) model of inflammation and colon cancer. Methods: Hsp70 mutant mice (Hsp70.1(-/-)/70.3(-/-)) and wild type (WT) littermates received AOM and 3 cycles of DSS and were sacrificed 24 wks later. Tumors were graded for histology and immunostained for p53, APC, beta-catenin, Cox-2 and iNOS and sequenced for p53 mutations. Results: Elevated adenomas developed in 4/10 WT mice with no dysplasia in adjacent mucosa. In contrast, 7/8 Hsp70 KO mice developed chronic mucosal inflammation and multifocal areas of flat dysplasia and 4/8 progressed to invasive carcinomas arising in a background of flat dysplastic mucosa. These differences in the incidence of flat dysplasia and invasive cancers were significant (p<0.05). Nuclear p53 was stronger in Hsp70 KO tumors compared to WT tumors, and sequencing confirmed p53 mutations in 2/5 tumors from Hsp70(-/-) vs. 0/5 in WT mice. In Hsp70 WT tumors beta-catenin was predominantly nuclear, compared with membranous beta-catenin in Hsp70(-/-) tumors, suggesting that Hsp70 regulates beta-catenin in colonic tumorigenesis. Cox-2 and iNOS levels were increased in tumors from Hsp70(-/-) mice compared to Hsp70 wild type tumors. Conclusions: Hsp70-deleted mice treated with AOM/DSS develop flat invasive colonic tumors that mimic many histological and molecular features of UC colon cancer. This model will be useful to dissect the role of Hsp70 in IBD colon cancer.

PMID: 19005184 [PubMed - as supplied by publisher]

HLA-DM Mediates Epitope Selection by a "Compare-Exchange" Mechanism when a Potential Peptide Pool Is Available.

PLoS ONE. 2008;3(11):e3722

Authors: Ferrante A, Anderson MW, Klug CS, Gorski J

BACKGROUND: HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties. CONCLUSION/SIGNIFICANCE: This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a "compare-exchange" sorting algorithm on an available peptide pool. Such a "third party"-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.

PMID: 19005572 [PubMed - in process]

Equilibrium Refolding Transitions Driven by Trifluoroethanol and by Guanidine Hydrochloride Dilution Are Similar in GTPase Effector Domain: Implications to Sequence-Self-Association Paradigm.

Biochemistry. 2008 Nov 12;

Authors: Chugh J, Sharma S, Hosur RV

Protein folding transitions starting from a denatured state play crucial roles in deciding the final fate of a protein. A fundamental question in this regard is the role of the amino acid sequence of the protein. In this context, we have investigated here the equilibrium refolding to a partially folded state of the GTPase effector domain (GED) of dynamin driven by addition of increasing amounts of trifluoroethanol (TFE) and compared it with that driven by progressive dilution of the guanidine hydrochloride (Gdn-HCl) denaturant, which has been reported recently [ Chugh et al. ( 2008) Protein Science 17, 1319- 1325 ]. The structural and dynamics changes as the molecule refolds starting from the Gdn-HCl denatured state have been monitored by circular dichroism, fluorescence, and NMR. The molecule remains a monomer in the TFE limiting case, whereas in the Gdn-HCl case, the molecule self-associates as the denaturant is removed. Even so, the two equilibrium transitions seem to have many similarities. The limiting helical contents are similar, and the regions of progressive increase in millisecond time scale motions, suggestive of slow conformational transitions, are largely the same. Though in the guanidine dilution case the partially folded molecules self-associate and there is multimer-monomer equilibrium, the very high concentration ( approximately 6 M) of guanidine prevents self-association in the case of TFE created species. Taken together, the observations under the drastically different solvation conditions suggest that the GED sequence is designed to self-assemble via helices leading to formation of a fully folded megadalton size assembly. The present observations may also have implications for the folding and association mechanism of the protein. These are important from the point of view of dynamin function.

PMID: 19006333 [PubMed - as supplied by publisher]

[In vitro cross-linking of Escherichia coli tartrate dehydratase beta subunit]

Sheng Wu Gong Cheng Xue Bao. 2008 Aug;24(8):1485-9

Authors: Xu Y, Gao Y

To test the hypothesis that in vitro protein cross-linking could be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links, we amplified wild and Cys/Ser mutant genes with PCR technique from E. coli BL21 cells and subcloned them into expression plasmid pTrcHisC. Recombinant proteins, which were associated with formation of inclusion bodies induced by IPTG, were purified by immobilized metal affinity chromatography (IMAC) and refolded by dialysis. In thermal unfolding and oxidative refolding experiment, wild TtdB was proved to form cross-linked dimmers/oligomers as revealed by SDS-PAGE; cross-linking intensity was obviously weakened when the loading buffer contained the reducing agent dithiothreitol (DTT). The residual cross-linking was isopeptide bonds; no dimmers/oligomers were detected when the refolding and unfolding solution contained DTT. In addition, Cys/Ser point mutation abrogated its ability to cross-link into homodimers, which showed disulfide bonds could facilitate the following formation of isopeptide bonds.

PMID: 18998556 [PubMed - in process]

[Renaturation, purification and antigenicity identification of recombinant protein of Cysticercus cellulosae expressed in Escherichia coli]

Sheng Wu Gong Cheng Xue Bao. 2008 Aug;24(8):1490-5

Authors: Zhang S, Jia W, Luo X, Jing Z, Wu G, Zheng Y, Guo A, Cai X

To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.

PMID: 18998557 [PubMed - in process]

Dendritic Molecular Switch: Chiral Folding and Helicity Inversion.

J Am Chem Soc. 2008 Nov 11;

Authors: Jiang X, Lim YK, Zhang BJ, Opsitnick EA, Baik MH, Lee D

Appropriately designed chemical architectures can fold to adopt well-defined secondary structures without the need for structural motifs of biological origin. We have designed tris( N-salicylideneaniline)-based hyperbranched molecules that spontaneously collapse to compact three-blade propeller geometry of either ( P)- or ( M)-handedness. For a homologous series of compounds, a direct correlation was established between the absolute screw sense, either ( P)- or ( M)-, of this helical folding and the absolute configuration, either ( R)- or ( S)-, of the chiral alcohol groups introducing local asymmetric bias to the conformationally restricted molecular backbone. (1)H NMR and CD spectroscopic studies provided significant insights into structural folding and unfolding of these chiral molecules in solution, which proceed via reversible assembly and disassembly of the C 3-symmetric hydrogen-bonding network. Notably, solvents profoundly influenced this dynamic process. A strong correlation between the solvent donor number (DN) or solvent basicity (SB) parameters and the change in the Cotton effects pointed toward specific O-H...solvent interactions that drive structural unfolding and eventual refolding to apparently opposite helicity. This unusual chirality inversion process could also be induced by installation of chemical protecting groups that simulate specific solvent-solute interactions. Removal of this covalent mimic of the solvent shell restored the original screw sense of the parent molecule, thus establishing the feasibility of covalently triggered helicity inversion as a new mode of operation for chiroptical molecular switches.

PMID: 18998685 [PubMed - as supplied by publisher]

Refolding of fully reduced bovine pancreatic trypsin.

J Biosci Bioeng. 2008 Oct;106(4):345-9

Authors: Ohshima Y, Suzuki Y, Nakatani A, Nohara D

Refolding of bovine pancreatic trypsin was carried out. When starting with denatured S-S intact trypsin, the recovered activity attained was 95-100%. In contrast, the recovered activity after refolding denatured S-S reduced trypsin was considerably low compared with other proteases that have been worked with previously. Such low recovered activity was attributed to the small amount of fully reduced trypsin used as starting material for complete refolding. Taking this into account, a recovered activity of 86% could be achieved when using inhibitor-immobilized gels.

PMID: 19000609 [PubMed - in process]

Large-Scale Production of Major House Dust Mite Allergen Der f 2 Mutant (C8/119S) in Escherichia coli.

J Biosci Bioeng. 2008 Oct;106(4):387-92

Authors: Koyanagi S, Maeda T, Murakami T, Kawatsu K, Sugawara K, Miyatsu Y, Mizokami H

Hyposensitization, in which causative antigens of allergic diseases are injected, is the sole means of a radical cure for allergic diseases. Since the therapeutic allergens currently used are naturally extracted, producing preparations with a stable titer from such extracts is extremely difficult. There are several reports on the expression of recombinant mite allergens in Escherichia coli using inducers. The use of an inducer for industrial production will lead to high costs and, for therapeutic use, it must be removed in the purification process. C8/119S is a mutant of Der f 2, a major house dust mite allergen. The C8/119S gene was integrated downstream of the trp promoter to produce the expression plasmid (pWU11-C8/119S). Then, this expression plasmid was used to transform E. coli strain HB101 (pWU11-C8/119S/HB101). A recombinant E. coli pWU11-C8/119S/HB101 did not express C8/119S in a low-temperature culture (32 degrees C), but C8/119S was induced to a high level of expression in a high-temperature culture (37 degrees C). pWU11-C8/119S/HB101 proliferated when expression was induced by high temperature and an approximately 3-fold greater proliferation was obtained compared with the use of an inducer in a large-scale culture. The C8/119S protein was expressed as inclusion bodies and obtained by refolding and chromatography purifications. The immunological properties of C8/119S were assessed by western blotting. Western blotting demonstrated that purified C8/119S reacted with a monoclonal anti-Der f 2 antibody (18G8). pWU11-C8/119S/HB101 can be used as an easy, low cost expression system on a large scale. It is also advantageous for industrial production in that the addition of an inducer is not required to achieve expression of the mite allergen.

PMID: 19000616 [PubMed - in process]

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate.

Extremophiles. 2008 Nov 12;

Authors: Ge M, Mao YJ, Pan XM

The alpha/beta-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other alpha/beta-mixed or pure beta-sheet proteins are compared.

PMID: 19002648 [PubMed - as supplied by publisher]

Related Articles

Aromatic Residues Engineered into the beta-Turn Nucleation Site of Ubiquitin Lead to a Complex Folding Landscape, Non-Native Side-Chain Interactions, and Kinetic Traps.

Biochemistry. 2008 Nov 8;

Authors: Rea AM, Simpson ER, Meldrum JK, Williams HE, Searle MS

The fast folding of small proteins is likely to be the product of evolutionary pressures that balance the search for native-like contacts in the transition state with the minimum number of stable non-native interactions that could lead to partially folded states prone to aggregation and amyloid formation. We have investigated the effects of non-native interactions on the folding landscape of yeast ubiquitin by introducing aromatic substitutions into the beta-turn region of the N-terminal beta-hairpin, using both the native G-bulged type I turn sequence (T XTGK) as well as an engineered 2:2 XNGK type I' turn sequence. The N-terminal beta-hairpin is a recognized folding nucleation site in ubiquitin. The folding kinetics for wt-Ub (TLTGK) and the type I' turn mutant (TNGK) reveal only a weakly populated intermediate, however, substitution with X = Phe or Trp in either context results in a high propensity to form a stable compact intermediate where the initial U-->I collapse is visible as a distinct kinetic phase. The introduction of Trp into either of the two host turn sequences results in either complex multiphase kinetics with the possibility of parallel folding pathways, or formation of a highly compact I-state stabilized by non-native interactions that must unfold before refolding. Sequence substitutions with aromatic residues within a localized beta-turn capable of forming non-native hydrophobic contacts in both the native state and partially folded states has the undesirable consequence that folding is frustrated by the formation of stable compact intermediates that evolutionary pressures at the sequence level may have largely eliminated.

PMID: 18991391 [PubMed - as supplied by publisher]

Related Articles

Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA.

Biochemistry. 2008 Nov 8;

Authors: Sanchez KM, Gable JE, Schlamadinger DE, Kim JE

Refolding curves of the integral membrane protein outer membrane protein A (OmpA) were measured to determine the conformational stabilities of this model system for membrane protein folding. Wild-type OmpA exhibits a free energy of unfolding ( Delta G H 2 O degrees ) of 10.5 kcal/mol. Mutants, containing a single tryptophan residue at the native positions 7, 15, 57, 102, or 143, are less stable than wild-type OmpA, with Delta G H 2 O degrees values of 6.7, 4.8, 2.4, 4.7, and 2.8 kcal/mol, respectively. The trend observed here is discussed in terms of noncovalent interactions, including aromatic interactions and hydrogen bonding. The effect of the soluble tail on the conformational stability of the transmembrane domain of OmpA was also investigated via truncated single-Trp mutants; Delta G H 2 O degrees values for four of the five truncated mutants are greater by >2.7 kcal/mol relative to the full-length versions, suggesting that the absence of the soluble domain may destabilize the unfolded transmembrane domain. Finally, dynamic light scattering experiments were performed to measure the effects of urea and protein on vesicle size and stability. Urea concentrations greater than 1 M cause an increase in vesicle size, and these diameters are unaltered in the presence of protein. These dynamic light scattering results complement the fluorescence studies and illustrate the important effects of vesicle size on protein conformational stability.

PMID: 18991402 [PubMed - as supplied by publisher]

Novobiocin and Additional Inhibitors of the Hsp90 C-Terminal Nucleotide-binding Pocket.

Curr Med Chem. 2008;15(26):2702-17

Authors: Donnelly A, Blagg BS

The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site ( approximately 700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site. EGCG is one of the active ingredients found in green tea. EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding. Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimics the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones, Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis. This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity.

PMID: 18991631 [PubMed - in process]

Equilibrium folding of porcine insulin precursor in the presence of redox buffer: implications for the common intermediates shared by its unfolding/ refolding processes.

Protein Pept Lett. 2008;15(9):972-9

Authors: Zhao J, Huang QL, Tang YH, Guo ZY, Qiao ZS, Xu GJ, Feng YM

We use the procedure established for 'disulfide stability analysis in redox system' to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form. Besides, the comparison of the intermediates captured in PIP unfolding process with those intermediates captured in its refolding process revealed that some intermediates captured during both unfolding/refolding processes of PIP have identical disulfide pairing pattern, from which we suggest that the unfolding/refolding processes of PIP share some common intermediates but flow in the opposite direction.

PMID: 18991774 [PubMed - in process]

Strategies for recombinant expression of small, highly disulphide-bonded, cationic antimicrobial peptides.

Protein Pept Lett. 2008;15(9):985-94

Authors: Greenshields AL, Knickle LC, Syvitski R, Douglas SE

Expression of two recombinant hepcidin homologues from Atlantic salmon, Salmo salar, characterization of their antimicrobial activity, and partial structural determination of the peptides is described. Expression was attempted in baculovirus and bacterial expression systems and the various purification and refolding methods used to determine the optimal strategy for production of active, correctly refolded hepcidin are reviewed.

PMID: 18991776 [PubMed - in process]

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Kinetic and Structural Studies of Phosphodiesterase-8A and Implication on the Inhibitor Selectivity.

Biochemistry. 2008 Nov 5;

Authors: Wang H, Yan Z, Yang S, Cai J, Robinson H, Ke H

Cyclic nucleotide phosphodiesterase-8 (PDE8) is a family of cAMP-specific enzymes and plays important roles in many biological processes, including T-cell activation, testosterone production, adrenocortical hyperplasia, and thyroid function. However, no PDE8 selective inhibitors are available for trial treatment of human diseases. Here we report kinetic properties of the highly active PDE8A1 catalytic domain prepared from refolding and its crystal structures in the unliganded and 3-isobutyl-1-methylxanthine (IBMX) bound forms at 1.9 and 2.1 A resolutions, respectively. The PDE8A1 catalytic domain has a K M of 1.8 muM, V max of 6.1 mumol/min/mg, a k cat of 4.0 s (-1) for cAMP, and a K M of 1.6 mM, V max of 2.5 mumol/min/mg, a k cat of 1.6 s (-1) for cGMP, thus indicating that the substrate specificity of PDE8 is dominated by K M. The structure of the PDE8A1 catalytic domain has similar topology as those of other PDE families but contains two extra helices around Asn685-Thr710. Since this fragment is distant from the active site of the enzyme, its impact on the catalysis is unclear. The PDE8A1 catalytic domain is insensitive to the IBMX inhibition (IC 50 = 700 muM). The unfavorable interaction of IBMX in the PDE8A1-IBMX structure suggests an important role of Tyr748 in the inhibitor binding. Indeed, the mutation of Tyr748 to phenylalanine increases the PDE8A1 sensitivity to several nonselective or family selective PDE inhibitors. Thus, the structural and mutagenesis studies provide not only insight into the enzymatic properties but also guidelines for design of PDE8 selective inhibitors.

PMID: 18983167 [PubMed - as supplied by publisher]

Related Articles

Refolding and kinetic characterization of the phosphodiesterase-8A catalytic domain.

Protein Expr Purif. 2008 Oct 19;

Authors: Yan Z, Wang H, Cai J, Ke H

Cyclic nucleotide phosphodiesterase-8 (PDE8) hydrolyzes the second messenger cAMP and is involved in many biological processes such as testosterone production. Although the bacterial and mammalian expression systems have been extensively tried, production of large quantity of soluble and active PDE8 remains to be a major hurdle for pharmacological and structural studies. Reported here is a detailed protocol of refolding and purification of large quantity of the PDE8A1 catalytic domain (residues 480-820) and kinetic characterization of the refolded protein. This protocol yielded about 8mg of the PDE8A catalytic domain from 2l Escherichia coli culture, which has at least 40-fold higher activity than those reported in literature. The PDE8A1 catalytic domain has k(cat) of 4.0s(-1) for Mn(2+) and 2.9s(-1) for Mg(2+), and the K(M) values of 1-1.8muM. In addition, the PDE8A1 (205-820) fragment that contains both PAS and catalytic domains was expressed in E. coli and refolded. This PDE8A1 (205-820) fragment has k(cat) of 1.1s(-1) and K(M) of 0.28muM, but aggregated at high concentration. The K(M) of PDE8A1 (205-820) is 2- to 7-fold higher than the K(M) values of 40-150nM for the full-length PDE8s in literature, but about 6-fold lower than that of the catalytic domain. Thus, the K(M) difference likely implies an allosteric regulation of the PDE8A activity by its PAS domain.

PMID: 18984055 [PubMed - as supplied by publisher]

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Small molecule inhibition of a Group II chaperonin: Pinpointing a loop region within the equatorial domain as necessary for protein refolding.

Arch Biochem Biophys. 2008 Oct 19;

Authors: Bergeron LM, Shis DL, Gomez L, Clark DS

The functionality of regions within the equatorial domain of Group II chaperonins is poorly understood. Previously we showed that a 70 amino acid sequence within this domain on the single-subunit recombinant thermosome from Methanocaldococcus jannaschii (rTHS) contains residues directly responsible for refolding protein substrates [L.M. Bergeron, C. Lee, D.S. Clark, Identification of a critical chaperoning region on an archaeal recombinant thermosome, Biochem. Biophys. Res. Commun. 369 (2008) 707-711]. In the present study, 6-aminopenicillanic acid (6-APA) was found to bind to rTHS and inhibit it from refolding proteins. Fluorescence anisotropy was used to measure a 6-APA/rTHS dissociation constant of 17.1muM and verify that the binding site is within the first 70 amino-terminal rTHS residues. Docking simulations point to a specific loop region at residues 53-57 on rTHS as the most likely binding region. This loop region is located within the oligomeric association sites of the wild-type thermosome. These results implicate a specific equatorial region of Group II chaperonins in the refolding of proteins, and suggest its importance in conformational changes that accompany chaperone function.

PMID: 18976628 [PubMed - as supplied by publisher]