Ammonium Sulfate Fractionation
1. To the DE52 eluate from Subheading 3.2., step 4 , add ammonium sulfate to 30 saturation 176 mg mL by slowly adding solid salt see Note 10 and stirring gently on ice. After complete addition, continue stirring for 10 min more. 2. Spin out precipitated proteins by centrifugation at 16,000g for 20 min. Keep supernatant and discard the pellet. 3. To the supernatant of step 2, slowly add ammonium sulfate to 50 of saturation 127 mg mL . After completing addition, stir gently on ice for 10 min. 4....
Electrophoresis 1
Screening of the fractions obtained by chromatography is carried out using preformed PHAST gels Pharmacia 12.5 for SDS gels and gradient 4-15 for native PAGE. Sample loading buffers are 5X SDS gel buffer 0.5 g SDS, 0.3 g DTT, and 1 mg pyronin Y dissolved in 10 mL 75 glycerol 25 50mM Tris-HCl, pH 7.5. 5X Native gel buffer 1 mg bromophenol blue dissolved in10 mL 75 glycerol 25 50mM Tris-HCl, pH 7.5. Alternatively, 12 Laemmli SDS gels 7 can be used for screening purposes. GroEL can also be...
Materials Oka
1. Strains To test chaperonin mutants and homolog proteins, we routinely use strains that have been derived from the widely used E. coli hosts TG1 and B178 by P1 transduction of the mutant chaperonin alleles as follows All strains are resistant to tetracycline. SV1, SV2, SV7, and SV8 are isogenic strains. E. coli strain B and B178 or SV1 are recommended for the growth of bacteriophages T4D0 and X b2cI , respectively. E. coli strain AI90 pBAD50 is the host strain for growing bacteriophage P1 and...
Preparative PAGE Separation of the Two Forms of the Archaeal Chaperonin
1. The purified chaperonin runs as a doublet in native gels. To separate the doublet, native one-dimensional 1D PAGE was optimized by examining a range of gel Fig. 3. Separation of the chaperonin from S. shibatae using native preparative PAGE A and subsequent 2D PAGE B and EM studies C and D . A Lane 1 pure chaperonin obtained from a freshly grown culture of S. shibatae 20 g of pure protein applied Lane 2 purified bottom band BB closed complex . Lane 3 purified top band TB open complex ....
Introduction Xox
The Escherichia coli chaperonin GroEL and its cochaperone GroES facilitate protein folding in an ATP-dependent reaction cycle for review, see 1,2 . GroEL is a cylindrical complex formed by two 7-mer-toroids, which are stacked back to back 3,4 . Both toroids describe a central cavity in which denatured proteins are bound in a molten globule state. The bound substrate is sequestered inside the cavity after GroES, a dome-shaped seven-membered ring 5 , closes the toroid in a cis-complex. Binding of...
Preparation of P pastoris Cytosol
1. Tris-dithiothreitol DTT buffer 0.1 M Tris-SO4, pH 9.4, 10 mM DTT. Prepare fresh just before use. 2. Sorbitol buffer 1.2 M sorbitol Baker Inc. Phillipsburg, NJ , 20 mM potassium phosphate, pH 7.4. 3. Breaking buffer 0.6 M sorbitol, 20 mM potassium phosphate, pH 7.4, 1 mM PMSF, protease inhibitor mix leupeptin 1.25 g mL , antipain 0.75 g mL , chymostatin 0.25 g mL , elastinal 0.25 g mL , pepstatin 5 g mL , phenylmethylsulfonyl fluoride 1 mM , and EDTA 1 mM see Note 1 . 4. Zymolyase ICN...
Info Hlg
Fig. 3. Comparison of thermophilic chaperonin with GroEL. For a detailed discussion, see Subheading 3.6. A Native gel of purified chaperonins. B Electron-micrographs of thermophilic chaperonins. C 2D gel patterns of the different chaperonins. D Comigration studies. The numbering scheme is as follows 1 T. litoralis, 2 S. shibatae, 3 T. thermophilus, 4 E. coli, 5 Pyrococcus furiosus. Fig. 3. Comparison of thermophilic chaperonin with GroEL. For a detailed discussion, see Subheading 3.6. A Native...
References Kkr
1. Georgopoulos, C. and Welch, W. J. 1993 Role of the major heat shock proteins as molecular chaperones. Ann. Rev. Cell Biol. 9, 601-634. 2. Landry, S. J. and Gierasch, Lila, M. 1994 Polypeptide interactions with molecular chaperones and their relationship to in vivo protein folding. Annu. Rev. Biophys. Biomol. Struct. 23, 645-649. 3. Hartl, F. U. and Martin, J. 1995 Molecular chaperones in cellular protein folding. Curr. Opinion Struct. Biol. 5, 92-102. 4. Srere, P. A. 1975 The enzymology of...
Notes Yrw
1. Siliconized MC tubes are used to prevent unspecific binding of MDH to tube walls and, hence, loss of material. 2. Several different buffers have been used in the literature, ranging from pH 7.2 to 7.8 12,16,25,26 . 3. The concentration of the chaperonins used may vary. 4. Two most common ATP-regenerating systems used in the literature are the creatine kinase phosphocreatine or pyruvate kinase phosphoenolpyruvate combination. Stocks for creatine kinase phosphocreatine ATP regenerating system...
Dissociation and Reassembly of GroEL Complexes
1. Buffer A 50 mM Tris-HCl, pH 7.5, 80 mM KCl, 1 mM dithiothreitol DTT , 0.1 mM ethylene diamine tetraacetic acid EDTA . 2. Disodium ATP A 100 mM solution in buffer A without DTT, stored in aliquots at -20 C see Note 4 . 3. Magnesium chloride stock solution 1 M in distilled water. 4. GroEL protein, purified as described in Chapter 3, stored in aliquots at -80 C. 5. 8 M urea solution in buffer A see Note 1 . 6. Gel-sizing desalting columns Sephadex G-50, 0.9 x 2.0 cm NICK columns, Pharmacia . 7....
Electrophoresis Qwb
Screening of the fractions obtained by chromatography is carried out using the PHAST system from Pharmacia. The preformed PHAST gels used are 20 for SDS gel electrophoresis. If the experimenter has no access to a PHAST system, the same result can be achieved using 20 Laemmli gels 5 running in any standard laboratory electrophoresis equipment. It may also be necessary to run native gels to ensure that the oligomer and not just the monomer is purified. In this case, adequate resolution between...
mMDH Refolding
For a 100 pL reaction, mix in 1.5 mL sMC tubes see Note 8 1 mg mL pyruvate kinase final 20 mg mL 0.5 M phosphoenolpyruvate final 10 mM Incubate at 37 C for at least 5 min. Transfer the respective 98 L mix to sMC tubes containing 50 M N-MDH final 1 M 2 L 50 yM D-MDH final 1 yM 2 2 2 L and simultaneously vortexing gently to mix throughly. At the zero time point, remove 5 L for immediate assay see below and or 10 L into an sMC tube containing 2 L 0.45 M CDTA quenches the chaperonin reaction...
Methods 31 Cell Growth
1. Approximately 1 L of media is placed in each of the 10 2.5-L flasks. The use of flasks with molded baffles increase aeration and allows faster cell growth. Two 250 mL flasks with 100 mL of media are also included for the overnight culture that serves as inocculum. 2. The flasks are autoclaved on the wet cycle for 20 min at 130 C, and the media removed and allowed to cool. The overproducing GroEL strain is ampicillin-resistant. Therefore, after cooling to room temperature, 1.6 mL of...
References Pbn
1. Reading, D. S., Hallberg, R. L., and Myers, A. M. 1989 Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor. Nature 337,655-659. 2. Viitanen, P. V., Lorimer, G. H., Seetharam, R., Gupta, R. S., Oppenheim, J., Thomas, J. O., et al. L992 Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring. J. Biol. Chem. 267, 695-698. 3. Dubaquie, Y., Looser, R., and Rospert, S. 1997 Significance of chaperonin10-mediated inhibition of ATP hydrolysis by...
What Is Nrd Protein
Malate dehydrogenase MDH is found in all eukaryotic cells as two isozymesimitochondrial mMDH and cytoplasmic soluble, sMDH 1 . In constrast, prokaryotes contain only a single form of MDH. The crystal structures of MDH from Escherichia coli 2,3 , porcine cytoplasm 4 , porcine mitochondria 5,6 , and Thermus flavus 7 have been solved and are essentially identical. Refer to ref. 8 for a comprehensive review on MDH. Pig heart mitochondial MDH has been extensively studied and has been the substrate...
Rhodanese Refolding Assay 1
1. Mix in a 1.5 mL microcentrifuge tube at 25 C 5 L of 1 M Na-thiosulfate freshly prepared . 5 L of 25 y,MGroES. giving final concentrations of 20 mM MOPS-NaOH, pH 7.2, 100 mM NaCl, 10 mM KCl, 5 mM Mg-acetate, 20 mM Na-thiosulfate containing 0.25 M GroEL and 0.5 M GroES see Notes 2 and 4 . 2. Add 247.5 L of this mixture to 2.5 L of denatured rhodanese final 0.25 M see Note 5 . Mix rapidly by vortexing or pipeting up and down, but avoid air bubbles see Note 6 . 3. To determine rhodanese activity...
Rhodanese Activity Assay
1. 1 M Potassium cyanide KCN . 2. 1 M Na-thiosulfate prepare fresh before use . 5. 15 Formaldehyde prepare fresh before use . 6. Ferric nitrate reagent prepare this reagent in a fume hood Slowly add 100 mL of 65 HNO3 to a stirring solution of 50 g Fe NO3 3 9H2O in 600 mL distilled water. Adjust to 750 mL, and filter the solution. The solution is stable for months.
Rhodanese Activity Assay 1
The amount of native rhodanese accumulated during the time-course of refolding is detected by enzyme activity assay see Note 7 . 1. Each 10 L aliquot of the refolding reaction, withdrawn at various timepoints, is immediately transferred to prepared microcentrifuge tubes containing the following assay mixture, preequilibrated at 25 C for 5 min 155 L distilled deionised water. A total volume of 200 L is obtained containing final concentrations of 50 mM KCN, 50 mM Na-thiosulfate, 40 mM KH2PO4, and...
Jorg Martin 1 Introduction
The assembly of chaperonin subunits into heptameric ring structures is a hallmark common to this group of proteins. In GroEL two of these rings are symmetrically stacked, resulting in a tetradecameric double-ring cylinder which is the functional form of the chaperonin. In this structure, unfolded substrate proteins bind to the apical subunit domains at the inner surface of the cylinder, whereas adenosine 5'-triphosphate ATP binding and hydrolysis occur at the level of the central subunit...
Electrophoresis
Screening of the fractions obtained by chromatography is carried out using preformed PHAST gels Pharmacia 12.5 for SDS gels and gradient 4-15 for native PAGE. The sample loading buffers are as follows 5X SDS 0.5 g SDS, 0.3 g DTT, and 1 mg pyronin Y dissolved in 10 mL 75 glycerol 25 50 mM Tris-HCl, pH 7.5. 5X Native 1 mg bromophenol blue dissolved in 10 mL 75 glycerol 25 50 mM Tris-HCl, pH 7.5. Alternatively, 12 Lammeli SDS gels 11 can be used for screening purposes. GroEL can also be detected...
Materials
1. E. coli strain MC1009 containing a plasmid that carries gene 31 from bacteriophage T4 under the transcriptional control of the inducible ara promoter 12,13 . 2. LB medium 10 g Bacto-tryptone, 5 g yeast extract, and 5 g NaCl L of H2O. Dissolve the reagents in 800 mL of H2O, and adjust the pH to 7.0 with 5 N NaOH. Make up to 1 L with H2O, and sterilize by autoclaving for 20 min at 15 lb sq. in. on a liquid cycle. 3. LB agar 1 w v agar in LB medium. Before autoclaving add the agar to the LB...
Unfolding and Refolding of GroES
1. Buffer A 50 mM Tris-HCl, pH 7.5, 100 mM NaCl. 2. GroES protein, purified as described in Chapter 4, and stored in aliquots at -80 C. 3.8 M urea solution in 50 mM Tris, pH 7.5 see Note 1 . 4. Dialysis cassettes Pierce, Rockford, IL , ready-to-use, with a capacity of 0.1-0.5 mL and a 3500-Da mol-wt cutoff see Note 2 . 5. Gel-sizing column Superdex 75 HR 10 30 Pharmacia, Uppsala, Sweden see Note 3 , and FPLC apparatus with fraction collector, UV-monitor 280 nm , and recorder. 6. Gel-filtration...