Reactivation of Aggregated Proteins by the ClpB/DnaK Bi‐Chaperone System

Michal Zolkiewski1, Liudmila S. Chesnokova2, Stephan N. Witt3

1 Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, 2 Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, 3 Department of Biochemistry and Molecular Biology and Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, Louisiana
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.10
DOI:  10.1002/0471140864.ps2810s83
Online Posting Date:  February, 2016
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Abstract

Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose‐6‐phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone, DnaK. The procedures for purification of these chaperones are also described. © 2016 by John Wiley & Sons, Inc.

Keywords: protein misfolding; protein aggregation; molecular chaperone; ClpB; DnaK

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Reactivation of Aggregated Glucose‐6‐Phosphate Dehydrogenase (G6PDH) in the Presence of ClpB and DnaK/DnaJ/GrpE
  • Support Protocol 1: Purification of E. coli ClpB
  • Support Protocol 2: Purification of E. coli DnaK
  • Support Protocol 3: Purification of E. coli GrpE
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Reactivation of Aggregated Glucose‐6‐Phosphate Dehydrogenase (G6PDH) in the Presence of ClpB and DnaK/DnaJ/GrpE

  Materials
  • 600 μM glucose‐6‐phosphate dehydrogenase (G6PDH) in 50 mM Tris·Cl, pH 7.5 (see appendix 2E for buffer); prepare using lyophilized G6PDH from Leuconostoc mesenteroides (Sigma‐Aldrich)
  • Denaturation buffer (see recipe)
  • Refolding buffer (see recipe)
  • Reactivation buffer (see recipe)
  • G6PDH assay buffer (see recipe)
  • Temperature‐controlled heat block or water bath
  • UV‐Vis spectrophotometer

Support Protocol 1: Purification of E. coli ClpB

  Materials
  • Expression plasmid for ClpB with IPTG‐dependent promoter (as described in Barnett et al., )
  • BL21(DE3) competent E. coli cells (available from Life Technologies and other vendors)
  • LB liquid medium and plates ( appendix 4A; Elbing and Brent, ) with 0.1 mg/ml ampicillin (or other antibiotic, depending on the expression plasmid)
  • Isopropyl β‐d‐1‐thiogalactopyranoside (IPTG)
  • SDS sample buffer (see unit 10.1; Gallagher, )
  • ClpB Buffers A, B, and C (see reciperecipes)
  • 80% (v/v) sterile glycerol
  • Polyethyleneimine
  • Potassium chloride (KCl)
  • Q Sepharose Fast Flow (GE Healthcare Life Sciences)
  • Ammonium sulfate
  • Superdex 200, prep grade (GE Healthcare Life Sciences)
  • Shaker‐incubator
  • Refrigerated centrifuge and centrifuge bottles
  • Balance
  • Probe sonicator
  • Glass spatula
  • 50 × 2.5–cm and 50 × 1.5–cm chromatography columns
  • Low‐pressure chromatography system with a gradient maker
  • 50 kDa MWCO dialysis membrane (also see appendix 3B; Zumstein, )
  • Concentrator (optional)
  • Additional reagents and equipment for transformation of E. coli ( appendix 4D; Seidman and Struhl, ), growth of E. coli ( appendix 4B;Elbing and Brent, ), SDS‐PAGE (unit 10.1; Gallagher, ) and gel staining (unit 10.5; Echan and Speicher, ), protein precipitation using ammonium sulfate ( appendix 3F; Wingfield, ), dialysis ( appendix 3B; Zumstein, ), and determination of protein concentration (unit 3.4; Olson and Markwell, )

Support Protocol 2: Purification of E. coli DnaK

  Materials
  • Expression vector: pMSK (Ampr) plasmid bearing dnaK gene under an IPTG promoter
  • E.coli BB1553 competent cells (ΔdnaK52::Cmr derivative of MC4100 with sidB1 secondary mutation; Bukau and Walker, ; these cells can be obtained form Bernd Bukau or almost any researchers in this field)
  • LB liquid medium and plates ( appendix 4A; Elbing and Brent, ) with 100 μg/ml ampicillin and 50 μg/ml chloramphenicol
  • Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • SDS‐PAGE loading buffer (unit 10.1; Gallagher, )
  • Pre‐cast 10% or 12% polyacrylamide gels (also see unit 10.1; Gallagher, )
  • 50% (w/w) glycerol, sterile
  • Buffer S: 50 mM Tris·Cl, pH 8.0 ( appendix 2E) containing 10% (w/v) sucrose
  • French Press Lysis Buffer (see recipe)
  • PMSF stock solution: dissolve 3 to 4 mg of phenylmethylsulfonyl fluoride (PMSF) in 1 ml dimethylsulfoxide (DMSO); prepare fresh
  • Ammonium sulfate, saturated, ice cold
  • DnaK Buffers A and B (see reciperecipes)
  • ATP‐agarose (Sigma, cat. no. A2767)
  • 6 mM ATP‐MgCl 2 (see recipe)
  • MONO Q low‐salt and high‐salt buffers (see reciperecipes)
  • Protein assay kit (Bio‐Rad)
  • Bovine gamma globulin (Bio‐Rad, standard I, cat. no. 500‐0005) as standard for protein assay
  • Glycerol
  • UV‐Vis spectrometer
  • Temperature‐controlled heat block or water bath
  • High‐speed refrigerated centrifuge (e.g., Beckman JA‐21 with J‐14 rotor)
  • French Press (Newport Scientific)
  • Sorvall centrifuge with SS‐34 rotor and Sorvall centrifuge tubes
  • MWCO 10 kDa (consistent with units in ClpB section) dialysis tubing
  • 50‐ml conical tubes (e.g., Corning Falcon)
  • 1 × 7–cm chromatography column
  • UV‐Vis spectrophotometer
  • MONO Q GL column (GE Healthcare Life Sciences, cat. no. 17‐5166‐01)
  • High‐performance liquid chromatography (HPLC) or fast protein liquid chromatography (FPLC system)
  • Additional reagents and equipment for transformation of E. coli ( appendix 4D; Seidman and Struhl, ), growth of E. coli including monitoring bacterial concentration by optical density (Elbing and Brent, ), SDS‐PAGE (Gallagher, ) and gel staining (unit 10.5; Echan and Speicher, ), protein precipitation using ammonium sulfate ( appendix 3F; Wingfield, ), and dialysis ( appendix 3B; Zumstein, )

Support Protocol 3: Purification of E. coli GrpE

  Materials
  • Expression vector: pWKG20 (Ampr) plasmid bearing grpE gene under an arabinose P BAD promoter (a generous gift from Dr. William L. Kelley (University of Geneva, Division of Infectious Diseases)
  • E.coli DA259 competent cells (C600 ΔgrpE : : ΩCmR thr : : Tn10; constructed by C. Georgopoulos and described in Sugimoto et al., )
  • LB liquid medium and plates ( appendix 4A; Elbing and Brent, ) with 100 μg/ml ampicillin and 50 μg/ml chloramphenicol
  • L‐arabinose
  • SDS‐PAGE loading buffer (unit 10.1; Gallagher, )
  • Pre‐cast 10% or 12% polyacrylamide gels (also see unit 10.1; Gallagher, )
  • 50% (w/w) glycerol, sterile
  • Buffer S: 50 mM Tris·Cl, pH 8.0 ( appendix 2E) containing 10% (w/v) sucrose
  • French Press Lysis Buffer (see recipe)
  • PMSF stock solution: dissolve 3 to 4 mg of phenylmethylsulfonyl fluoride (PMSF) in 1 ml dimethylsulfoxide (DMSO); prepare fresh
  • Ammonium sulfate
  • GrpE Buffers B and C (see reciperecipes)
  • Affi‐Gel 10 resin (Bio‐Rad, cat. no. 153‐6099)
  • Purified DnaK ( protocol 3)
  • KCl
  • ATP
  • MgCl 2
  • MONO Q low‐salt and high‐salt buffers (see reciperecipes)
  • Protein assay kit (Bio‐Rad)
  • Bovine gamma globulin (Bio‐Rad, standard I, cat. no. 500‐0005) as standard for protein assay
  • Glycerol
  • UV‐Vis spectrophotometer
  • Temperature‐controlled heat block or water bath
  • High‐speed refrigerated centrifuge (e.g., Beckman JA‐21 with J‐14 rotor)
  • French Press (Newport Scientific)
  • Sorvall centrifuge with SS‐34 rotor and Sorvall centrifuge tubes
  • MWCO 10 kDa dialysis tubing
  • Chromatography column (length = 13.5 cm; diameter = 2.5 cm)
  • UV‐Vis spectrophotometer
  • MONO Q GL column (GE Healthcare Life Sciences, cat. no. 17‐5166‐01)
  • High‐performance liquid chromatography (HPLC) or fast protein liquid chromatography (FPLC system)
  • Additional reagents and equipment for transformation of E. coli ( appendix 4D; Seidman and Struhl, ), growth of E. coli including monitoring bacterial concentration by optical density (Elbing and Brent, ), SDS‐PAGE (Gallagher, ) and gel staining (unit 10.5; Echan and Speicher, ), protein precipitation using ammonium sulfate ( appendix 3F; Wingfield, ), and dialysis ( appendix 3B; Zumstein, )
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Figures

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Literature Cited

Literature Cited
  Barnett, M.E., Zolkiewska, A., and Zolkiewski, M. 2000. Structure and activity of ClpB from Escherichia coli. Role of the amino‐and ‐carboxyl‐terminal domains. J. Biol. Chem. 275:37565‐37571. doi: 10.1074/jbc.M005211200.
  Barnett, M.E., Nagy, M., Kedzierska, S., and Zolkiewski, M. 2005. The amino‐terminal domain of ClpB supports binding to strongly aggregated proteins. J. Biol. Chem. 280:34940‐34945. doi: 10.1074/jbc.M505653200.
  Bosl, B., Grimminger, V., and Walter, S. 2006. The molecular chaperone Hsp104‐a molecular machine for protein disaggregation. J. Struct. Biol. 156:139‐148. doi: 10.1016/j.jsb.2006.02.004.
  Bukau, B. and Walker, G.C. 1990. Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone. EMBO J. 9:027‐036.
  Bukau, B., Weissman, J., and Horwich, A. 2006. Molecular chaperones and protein quality control. Cell 125:443‐451. doi: 10.1016/j.cell.2006.04.014.
  Cegielska, A. and Georgopoulos, C. 1989. Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis. J. Biol. Chem. 264:21122‐21130.
  de Marco, A., Deuerling, E., Mogk, A., Tomoyasu, T., and Bukau, B. 2007. Chaperone‐based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol. 7:32. doi: 10.1186/1472‐6750‐7‐32.
  Dean, D.O. and James, R. 1991. Identification of a gene, closely linked to dnaK, which is required for high‐temperature growth of Escherichia coli. J. Gen. Microbiol. 137:1271‐1277. doi: 10.1099/00221287‐137‐6‐1271.
  Desantis, M.E. and Shorter, J. 2012. The elusive middle domain of Hsp104 and ClpB: Location and function. Biochim. Biophys. Acta 1823:29‐39. doi: 10.1016/j.bbamcr.2011.07.014.
  Diamant, S., Ben‐Zvi, A.P., Bukau, B., and Goloubinoff, P. 2000. Size‐dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery. J. Biol. Chem. 275:21107‐21113. doi: 10.1074/jbc.M001293200.
  Doyle, S.M. and Wickner, S. 2009. Hsp104 and ClpB: Protein disaggregating machines. Trends Biochem. Sci. 34:40‐48. doi: 10.1016/j.tibs.2008.09.010.
  Echan, L.A. and Speicher, D.W. 2002. Protein detection in gels using fixation. Curr. Protoc. Protein Sci. 29:10.5.1-10.5.18.
  Elbing, K.L. and Brent, R. 1998a. Media preparation and bacteriological tools. Curr. Protoc. Protein Sci. 13:A.4A.1‐A.4A.5.
  Elbing, K. L. and Brent, R. 1998b. Growth in liquid or solid media. Curr. Protoc. Protein Sci. 13:A.4B.1‐A.4B.3.
  Fink, A.L. 1998. Protein aggregation: Folding aggregates, inclusion bodies and amyloid. Fold. Des. 3:R9‐23. doi: 10.1016/S1359‐0278(98)00002‐9.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Protein Sci. 68:10.1.1‐10.1.44.
  Glover, J.R. and Lindquist, S. 1998. Hsp104, Hsp70, and Hsp40: A novel chaperone system that rescues previously aggregated proteins. Cell 94:73‐82. doi: 10.1016/S0092‐8674(00)81223‐4.
  Goloubinoff, P., Mogk, A., Zvi, A.P., Tomoyasu, T., and Bukau, B. 1999. Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc. Natl. Acad. Sci. U.S.A. 96:13732‐13737. doi: 10.1073/pnas.96.24.13732.
  Grimminger, V., Richter, K., Imhof, A., Buchner, J., and Walter, S. 2004. The prion curing agent guanidinium chloride specifically inhibits ATP hydrolysis by Hsp104. J. Biol. Chem. 279:7378‐7383. doi: 10.1074/jbc.M312403200.
  Hartl, F.U., Bracher, A., and Hayer‐Hartl, M. 2011. Molecular chaperones in protein folding and proteostasis. Nature 475:324‐332. doi: 10.1038/nature10317.
  Hess, H.H. and Derr, J.E. 1975. Assay of inorganic and organic phosphorus in the 0.1‐5 nanomole range. Anal. Biochem. 63:607‐613. doi: 10.1016/0003‐2697(75)90388‐7.
  Horwich, A. 2002. Protein aggregation in disease: A role for folding intermediates forming specific multimeric interactions. J. Clin. Invest. 110:1221‐1232. doi: 10.1172/JCI0216781.
  Kedzierska, S. and Matuszewska, E. 2001. The effect of co‐overproduction of DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat‐aggregated proteins from Escherichia coli DeltaclpB mutant cells‐new insight into the role of Hsp70 in a functional cooperation with Hsp100. FEMS Microbiol. Lett. 204:355‐360.
  Lanzetta, P.A., Alvarez, L.J., Reinach, P.S., and Candia, O.A. 1979. An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100:95‐97. doi: 10.1016/0003‐2697(79)90115‐5.
  Miot, M., Reidy, M., Doyle, S.M., Hoskins, J.R., Johnston, D.M., Genest, O., Vitery, M.C., Masison, D.C., and Wickner, S. 2011. Species‐specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation. Proc. Natl. Acad. Sci. U.S.A. 108:6915‐6920. doi: 10.1073/pnas.1102828108.
  Montgomery, D.L., Morimoto, R.I., and Gierasch, L.M. 1999. Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling. J. Mol. Biol. 286:915‐932. doi: 10.1006/jmbi.1998.2514.
  Motohashi, K., Watanabe, Y., Yohda, M., and Yoshida, M. 1999. Heat‐inactivated proteins are rescued by the DnaK.J‐GrpE set and ClpB chaperones. Proc. Natl. Acad. Sci. U.S.A. 96:7184‐7189. doi: 10.1073/pnas.96.13.7184.
  Nagy, M., Wu, H.C., Liu, Z., Kedzierska‐Mieszkowska, S., and Zolkiewski, M. 2009. Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB. Protein Sci. 18:287‐293. doi: 10.1002/pro.36.
  Nagy, M., Guenther, I., Akoyev, V., Barnett, M.E., Zavodszky, M.I., Kedzierska‐Mieszkowska, S., and Zolkiewski, M. 2010. Synergistic cooperation between two ClpB isoforms in aggregate reactivation. J. Mol. Biol. 396:697‐707. doi: 10.1016/j.jmb.2009.11.059.
  Neuwald, A.F., Aravind, L., Spouge, J.L., and Koonin, E.V. 1999. AAA+: A class of chaperone‐like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9:27‐43.
  Rosenzweig, R., Moradi, S., Zarrine‐Afsar, A., Glover, J.R., and Kay, L.E. 2013. Unraveling the mechanism of protein disaggregation through a ClpB‐DnaK interaction. Science 339:1080‐1083. doi: 10.1126/science.1233066.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29.
  Rousseau, F., Schymkowitz, J., and Serrano, L. 2006. Protein aggregation and amyloidosis: Confusion of the kinds? Curr. Opin. Struct. Biol. 16:118‐126. doi: 10.1016/j.sbi.2006.01.011.
  Saibil, H. 2013. Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev. Mol. Cell Biol. 14:630‐642. doi: 10.1038/nrm3658.
  Sambrook, J.F.E. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Seidman, C.E. and Struhl, K. 1998. Introduction of plasmid DNA into cells. Curr. Protoc. Protein Sci. 13:A.4D.1‐A.4D.2.
  Squires, C.L., Pedersen, S., Ross, B.M., and Squires, C. 1991. ClpB is the Escherichia coli heat shock protein F84.1. J. Bacteriol. 173:4254‐4262.
  Sugimoto, S., Saruwatari, K., Higashi, C., and Sonomoto, K. 2008. The proper ratio of GrpE to DnaK is important for protein quality control by the DnaK‐DnaJ‐GrpE chaperone system and for cell division. Microbiology 154:1876‐1885. doi: 10.1099/mic.0.2008/017376‐0.
  Valastyan, J.S. and Lindquist, S. 2014. Mechanisms of protein‐folding diseases at a glance. Dis. Model. Mech. 7:9‐14. doi: 10.1242/dmm.013474.
  Wingfield, P. 1998. Protein precipitation using ammonium sulfate. Curr. Protoc. Protein Sci. 13:A.3F.1-A.3F.8.
  Woo, K.M., Kim, K.I., Goldberg, A.L., Ha, D.B., and Chung, C.H. 1992. The heat‐shock protein ClpB in Escherichia coli is a protein‐activated ATPase. J. Biol. Chem. 267:20429‐20434.
  Zhang, T., Ploetz, E.A., Nagy, M., Doyle, S.M., Wickner, S., Smith, P.E., and Zolkiewski, M. 2012. Flexible connection of the N‐terminal domain in ClpB modulates substrate binding and the aggregate reactivation efficiency. Proteins 80:2758‐2768. doi: 10.1002/prot.24159.
  Zolkiewski, M. 1999. ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi‐chaperone system from Escherichia coli. J. Biol. Chem. 274:28083‐28086. doi: 10.1074/jbc.274.40.28083.
  Zolkiewski, M., Zhang, T., and Nagy, M. 2012. Aggregate reactivation mediated by the Hsp100 chaperones. Arch. Biochem. Biophys. 520:1‐6. doi: 10.1016/j.abb.2012.01.012.
  Zumstein, L. 1995. Dialysis. Curr. Protoc. Protein Sci. 00:A.3B.1‐A.3B.4.
  Zylicz, M., Ang, D., and Georgopoulos, C. 1987. The grpE protein of Escherichia coli. purification and properties. J. Biol. Chem. 262:17437‐17442.
  Zylicz, M., Ang, D., Liberek, K., and Georgopoulos, C. 1989. Initiation of lambda DNA replication with purified host‐ and bacteriophage‐encoded proteins: The role of the dnaK, dnaJ and grpE heat shock proteins. EMBO J. 8:1601‐1608.
  Zylicz, M., Yamamoto, T., McKittrick, N., Sell, S., and Georgopoulos, C. 1985. Purification and properties of the dnaJ replication protein of Escherichia coli. J. Biol. Chem. 260:7591‐7598.
Key References
  Glover and Lindquist, 1998. See above.
  The first in vitro reconstitution of the protein disaggregating system of yeast chaperones.
  Goloubinoff et al., 1999. See above.
  The first description of the bacterial protein disaggregating system of chaperones.
  Motohashi et al., 1999. See above.
  The first description of the bacterial protein disaggregating system of chaperones.
  Rosenzweig et al., 2013. See above.
  Demonstration of a direct interaction between ClpB and DnaK.
  Zolkiewski, M. 1999. See above.
  The first description of the bacterial protein disaggregating system of chaperones.
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