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• W2110829900 abstract "Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation. Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation. Protein folding and maintenance in the cell require several molecular chaperones, which form transient, dynamic complexes with their protein substrates through hydrophobic interactions (1Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (916) Google Scholar, 2Bukau B. Deuerling E. Pfund C. Craig E. Cell. 2000; 101: 119-122Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Chaperone proteins from several families specialize in performing particular functions; therefore cooperation between chaperones from different families is very often required (1Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (916) Google Scholar, 2Bukau B. Deuerling E. Pfund C. Craig E. Cell. 2000; 101: 119-122Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Research in many laboratories has defined the function of chaperones within individual families. Yet the mechanistic details of the cooperation among the chaperones of different families are much less understood. ClpB, a member of the Hsp100 family in Escherichia coli, was identified as a thermotolerance conferring factor because mutations in the clpB gene severely decrease the survival rate of bacteria exposed to extreme heat (3Squires C.L. Pendersen S. Ross B. Squires C. J. Bacteriol. 1991; 173: 4254-4262Crossref PubMed Google Scholar, 4Kitagawa M. Wada C. Yoshioka S. Yura T. J. Bacteriol. 1991; 173: 4247-4253Crossref PubMed Google Scholar). In yeast Saccharomyces cerevisiae a similar decrease was observed in the survival rate of the hsp104 deletion strain (5Sanchez Y. Lindquist S. Science. 1990; 248: 1112-1115Crossref PubMed Scopus (656) Google Scholar, 6Sanchez Y. Taulien J. Borkovich K.A. Lindquist S. EMBO J. 1992; 11: 2357-2364Crossref PubMed Scopus (469) Google Scholar), which lacks the cytosolic member of the Hsp100 family. It was also shown that Hsp78, a mitochondrial member of the same chaperone family, plays a role in the thermotolerance of the respiratory function of mitochondria (7Schmitt M. Neupert W. Langer T. J. Cell Biol. 1996; 134: 1375-1386Crossref PubMed Scopus (80) Google Scholar). Heat treatment of both bacteria and yeast results in the accumulation of aggregated proteins inside cells (8Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 373: 475-478Crossref Scopus (737) Google Scholar, 9Laskowska E. Kuczynska-Wisnik D. Skórko-Glonek J. Taylor A. Mol. Microbiol. 1996; 22: 555-571Crossref PubMed Scopus (113) Google Scholar, 10Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (516) Google Scholar). Consistent with the above results, these aggregates are not eliminated in clpB or hsp104 deletion strains (8Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 373: 475-478Crossref Scopus (737) Google Scholar, 9Laskowska E. Kuczynska-Wisnik D. Skórko-Glonek J. Taylor A. Mol. Microbiol. 1996; 22: 555-571Crossref PubMed Scopus (113) Google Scholar, 10Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (516) Google Scholar). Studies on the reactivation of aggregated proteins following thermal stress showed that chaperones from the Hsp100 family alone are not sufficient for disaggregation. Other chaperone proteins are additionally involved in this process. For example, in E. coli Hsp70 (DnaK) and its co-chaperones cooperate with ClpB in the disaggregation of several aggregated proteins (10Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (516) Google Scholar). Similarly, in studies performed on yeast it was shown that besides Hsp78, mitochondrial Hsp70 and its co-chaperones are needed for protection and reactivation of the organelle functions following heat stress (7Schmitt M. Neupert W. Langer T. J. Cell Biol. 1996; 134: 1375-1386Crossref PubMed Scopus (80) Google Scholar, 11Germaniuk A. Liberek K. Marszalek J. J. Biol. Chem. 2002; 277: 27801-27808Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The E. coli Hsp100 (ClpB) protein belongs to the AAA+ (ATPase associated with various cellular activities) superfamily of proteins. AAA+ proteins self-assemble into oligomeric structures and use the energy from ATP hydrolysis to remodel their target substrates (12Vale R.D. J. Cell Biol. 2000; 150: F13-F19Crossref PubMed Google Scholar). ClpB consists of an N-terminal domain and two ATP-binding domains that mediate ATP hydrolysis, are essential for ClpB oligomerization and chaperone function (13Krzewska J. Konopa G. Liberek K. J. Mol. Biol. 2001; 314: 901-910Crossref PubMed Scopus (40) Google Scholar, 14Weizbezahn J. Schlieker C. Bukau B. Mogk A. J. Biol. Chem. 2003; 278: 32608-32617Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Schirmer E.C. Ware D.M. Queitsh C. Kowal A.S. Lindquist S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 914-919Crossref PubMed Scopus (74) Google Scholar, 16Watanabe Y. Motohashi K. Yoshida M. J. Biol. Chem. 2002; 277: 5804-5809Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Mogk A. Schlieker C. Strub C. Rist W. Weibezahn J. Bukau B. J. Biol. Chem. 2003; 278: 17615-17624Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), and are separated by a “linker” region (18Schirmer E.C. Glover J.R. Singer M.A. Lindquist S. Trends Biochem. Sci. 1996; 21: 289-296Abstract Full Text PDF PubMed Scopus (576) Google Scholar). Recent structural data obtained for ClpB from Thermus thermophilus show that the hexamer of this protein forms a two-tiered ring structure with a 16-Å hole in the top ring and six smaller openings on the lateral surface of the molecule (19Lee S. Sowa M.E. Watanabe Y.H. Sigler P.B. Yoshida M. Tsai F.T. Cell. 2003; 115: 229-240Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). The other group of chaperones required for efficient protein disaggregation, that is Hsp70 and its co-chaperones, has been studied for a relatively long time. The E. coli Hsp70 (DnaK) chaperone and its DnaJ and GrpE cohorts play a role in a variety of cellular processes including the folding of newly synthesized proteins (20Teter S.A. Houry W.A. Ang D. Tradler T. Rockabrand D. Fisher G. Blum P. Georgopoulos C. Hartl F-U. Cell. 1999; 97: 755-765Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 21Deuerling E. Schulze-Specking A. Tomayasu T. Mogk A. Bukau B. Nature. 1999; 400: 693-696Crossref PubMed Scopus (408) Google Scholar), as well as preventing protein aggregation under stress conditions (10Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (516) Google Scholar, 22Gragerov A. Nudler E. Komissarova N. Gaitanaris G. Gottesman M. Nikiforov V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10341-10344Crossref PubMed Scopus (184) Google Scholar, 23Hesterkamp T. Bukau B. EMBO J. 1998; 17: 4818-4828Crossref PubMed Scopus (103) Google Scholar). Correct selection of substrates is crucial for DnaK function. The affinity of DnaK for substrates is regulated by ATP, e.g. DnaK exhibits low affinity and a fast exchange rate for substrates in the ATP-bound state and high affinity and a low exchange rate in the ADP-bound state (24Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (423) Google Scholar). DnaJ triggers the hydrolysis of DnaK-bound ATP (25Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (690) Google Scholar) and thus modulates the affinity of DnaK for substrates. An additional role of DnaJ is to target substrates to DnaK (26Wawrzynow A. Zylicz M. J. Biol. Chem. 1995; 270: 19300-19306Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Liberek K. Wall D. Georgopoulos C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6224-6228Crossref PubMed Scopus (93) Google Scholar, 28Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (472) Google Scholar, 29Han W. Christen P. J. Biol. Chem. 2003; 278: 19038-19043Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Substrate release is mediated by ADP/ATP exchange, a process catalyzed by the GrpE co-chaperone, a nucleotide exchange factor. In some processes, the DnaK/DnaJ/GrpE chaperone system cooperates with other chaperones, for example with the Hsp60 (GroEL/GroES) system in protein folding (30Langer T. Lu C. Echlos H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (790) Google Scholar) and both Hsp100 (ClpB) (31Goloubinoff P. Mogk A. Zvi A.P.B. Tomayasu T. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13732-13737Crossref PubMed Scopus (502) Google Scholar, 32Motohashi K. Watanabe T. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (225) Google Scholar, 33Zolkiewski M. J. Biol. Chem. 1999; 274: 28083-28086Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar) and small heat shock proteins (IbpA and IbpB) (34Mogk A. Schlieker C. Friedrich K.L. Schönfeld H.-J. Vierling E. Bukau B. J. Biol. Chem. 2003; 278: 31033-31042Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) in the disaggregation of protein aggregates. The ability of the DnaK/DnaJ/GrpE system itself to reactivate aggregated proteins is limited to certain substrates, including RNA polymerase (35Skowyra D. Georgopoulos C. Zylicz M. Cell. 1990; 62: 939-944Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 36Blaszczak A. Zylicz M. Georgopoulos C. Liberek K. EMBO J. 1995; 14: 5085-5093Crossref PubMed Scopus (61) Google Scholar) and the DnaA protein (37Hwang D.S. Crooke E. Kornberg A. J. Biol. Chem. 1990; 265: 19244-19248Abstract Full Text PDF PubMed Google Scholar, 38Banecki B. Kaguni J.M. Marszalek J. Biochim. Biophys. Acta. 1998; 1442: 39-48Crossref PubMed Scopus (11) Google Scholar). The addition of ClpB protein to the DnaK/DnaJ/GrpE reactivation system results in a much broader range of potential substrates and furthermore lowers the concentration of chaperones needed for the efficient resolubilization of aggregated substrates in vitro (31Goloubinoff P. Mogk A. Zvi A.P.B. Tomayasu T. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13732-13737Crossref PubMed Scopus (502) Google Scholar, 32Motohashi K. Watanabe T. Yohda M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7184-7189Crossref PubMed Scopus (225) Google Scholar, 39Diamant S. Ben-Zvi A.P. Bukau B. Goloubinoff P. J. Biol. Chem. 2000; 275: 21107-21113Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Similar chaperone systems able to actively disaggregate denatured protein substrates in vitro were established for yeast cytosolic and mitochondrial chaperone proteins. Each of these systems, in analogy to the bacterial one, consists of a member of the Hsp100 family (Hsp104 for yeast cytosol and Hsp78 for yeast mitochondria) and an Hsp70 family member with its co-chaperones (Ssa1p or Ydj1p for yeast cytosol and Ssc1p, Mdj1p, or Mge1p for yeast mitochondria) (40Glover J.R. Lindquist S. Cell. 1998; 94: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar, 41Krzewska J. Langer T. Liberek K. FEBS Lett. 2001; 489: 92-96Crossref PubMed Scopus (123) Google Scholar). Attempts to replace the components of each system with the homologous chaperones from another cellular compartment or organism indicated that a specific functional cooperation exists between chaperones from the Hsp100 and Hsp70 families in the reactivation process (40Glover J.R. Lindquist S. Cell. 1998; 94: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar, 41Krzewska J. Langer T. Liberek K. FEBS Lett. 2001; 489: 92-96Crossref PubMed Scopus (123) Google Scholar, 42Schlee S. Beinker P. Akhrymuk A. Reinstein J. J. Mol. Biol. 2004; 336: 275-285Crossref PubMed Scopus (71) Google Scholar). However, the mechanism of this cooperation and the order of events leading to efficient disaggregation are unclear. We investigated in detail the requirements for the cooperation between ClpB and the DnaK/DnaJ/GrpE system in the disaggregation of aggregated substrate proteins. To do this, we developed a system that enabled us to follow real time measurements of the disaggregation reaction. Protein Purification—GFP 1The abbreviations used are: GFP, green fluorescent protein; ATPγS, adenosine 5′-O-(thiotriphosphate). was purified from E. coli DH5α transformed with the pGFPuv plasmid (Clontech). GFP overproduction was induced by isopropyl-β-d-thiogalactopyranoside addition. The cells were lysed in a French press (Aminco) at 1000 p.s.i. The bacterial lysate was centrifuged at 26,000 rpm for 30 min, and the supernatant was mixed on ice with 96% ethanol (1/1, v/v). Ethanol-precipitated proteins were sedimented at 28,000 rpm. Green fluorescent supernatant containing mostly GFP was immediately dialyzed against buffer containing 40 mm Tris-HCl, pH 7.5, 10% glycerol (v/v), 100 mm NaCl. After the addition of ammonium sulfate to 20% of saturation, the proteins were loaded on a Phenyl-Sepharose HR5/5 (Amersham Biosciences) column and eluted with a 100-ml 20–0% (percentage of saturation) ammonium sulfate and 0–20% (v/v) glycerol gradient. Fractions containing GFP were pooled and dialyzed to remove salt. GFP present in these fractions was concentrated by a step elution from a 2-ml Q-Sepharose Fast Flow (Amersham Biosciences) column with buffer containing 40 mm Tris-HCl, pH 7.5, 10% (v/v) glycerol, and 150 mm NaCl. Published protocols were used for the purification of E. coli DnaK, DnaJ, GrpE, (43Zylicz M. Ang D. Liberek K. Georgopoulos C. EMBO J. 1989; 8: 1601-1608Crossref PubMed Scopus (203) Google Scholar), and ClpB (44Woo K.M. Kim K.L. Goldberg A.L. Ha D.B. Chung C.H. J. Biol. Chem. 1992; 267: 20429-20434Abstract Full Text PDF PubMed Google Scholar). Firefly luciferase (E1701) was purchased from Promega. Protein concentrations were determined with the Bradford (Bio-Rad) assay system, using bovine serum albumin as a standard. Molar concentrations are given assuming a hexameric structure for ClpB and a monomeric structure for the rest of the proteins. GFP Disaggregation Experiments—GFP at a concentration of 2 mg/ml was thermally inactivated in buffer containing 40 mm Tris-HCl, pH 7.5, 10% (v/v) glycerol, and 150 mm NaCl by incubation for 15 min at 85 °C in 5-μl aliquots. Following thermal inactivation GFP was frozen in liquid nitrogen. Disaggregation reactions were performed at 25 °C in a 450-μl final volume in a spectrofluorometric cuvette in buffer containing 40 mm Tris-HCl, pH 7.8, 50 mm NaCl, 20 mm KCl, 20 mm MgCl2, 5 mm β-mercaptoethanol, and 10% (v/v) glycerol. In the experiments presented in Figs. 3 and 4B potassium glutamate was used at 85 mm concentration instead of KCl and NaCl. ATP was present in all reaction mixtures at 5 mm concentration. GFP in the disaggregation reaction was present at 0.5 μm, DnaK at 1 μm, DnaJ at 0.2 μm, GrpE at 0.1 μm, and ClpB at 0.65 μm concentration. Increase in GFP fluorescence was measured in a PerkinElmer Life Sciences LS50B spectrofluorometer with excitation at 395 nm and emission at 510 nm. The entrance slit was set to 5 nm, and the emission slit was set to 10 nm. The short time scale plots presented in Figs. 3B and 4 were corrected for the starting point to facilitate comparison. The fluorescence recorded at 510 nm was proportional to the amount of native GFP in the cuvette, which allowed us to calculate the rate of GFP reactivation.Fig. 4Both chaperones and nucleotides are required for the rapid initiation of fluorescence increase during GFP disaggregation. A, GFP aggregates were incubated with either DnaK, DnaJ, and GrpE; DnaK and DnaJ; DnaK; DnaJ; or no chaperones for 5 min. Following this, the missing chaperones were added, and the increase of fluorescence was monitored. B, aggregated GFP was incubated first with DnaK and DnaJ chaperones in the presence of 1 mm ATP, 1 mm ADP, or 1 mm ATPγS. After 5 min of incubation, ClpB and GrpE chaperones along with 10 mm ATP were added, and the increase of GFP fluorescence was monitored. GFP fluorescence was measured at 510 nm following excitation at 395 nm.View Large Image Figure ViewerDownload (PPT) Light Scattering Experiments—Light scattering experiments were essentially performed as GFP disaggregation experiments. Light scattering was measured in a PerkinElmer Life Sciences LS50B spectrofluorometer with the excitation and emission set at 310 nm. The entrance and emission slits were set to 5 nm. The solution of chaperones was additionally subjected to a short spin prior to the addition to cuvette. Sizing Chromatography Experiments—Native or heat-denatured GFP (20 μg) in buffer 40 mm Tris-HCl, pH 7.8, 100 mm NaCl was loaded onto a Sephacryl S-500 HR column (0.7 × 29 cm) equilibrated with the same buffer. Chromatography was carried out at a flow rate of 0.4 ml/min at 4 °C. The fractions (200 μl) were collected and analyzed by SDS-PAGE followed by Western blot analysis with anti-GFP monoclonal antibodies. The column was calibrated with the Bio-Rad gel filtration chromatography standards. The void volume of the column was determined by sizing the intact E. coli cells overproducing GFP followed by fluorescence analysis. Sedimentation Experiments—The reaction mixture contained 15 μg of native or heat-denatured GFP, and as indicated 10 μg of ClpB, 5 μg of DnaJ, and 10 μg of DnaK in 50 μl of buffer (40 mm Tris-HCl, pH 7.8, 50 mm NaCl, 20 mm KCl, 20 mm MgCl2, 5 mm β-mercaptoethanol, and 5mm ATP or ATPγS). The chaperone proteins and native GFP samples were briefly spun down prior to the start of the reaction. The reaction mixture was applied on a 3-ml 15–40% (v/v) glycerol gradient in the same buffer containing 0.5 mm ATP or ATPγS. The gradients were sedimented at 4 °C in a Beckman SW 60 rotor for 22 h at 46,000 rpm. The fractions were collected from the top of the gradients. The proteins present at the bottom of the centrifugation tube were resuspended in buffer containing 1% SDS, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Refolding of Guanidine Hydrochloride-denatured Luciferase—Firefly luciferase (2 μm) was denatured for 3 h at 37°Cin buffer containing 40 mm Tris-HCl, pH 7.4, 50 mm KCl, 1 mm dithiothreitol, 15 mm magnesium acetate, 5% (v/v) glycerol, and 2 m guanidine hydrochloride. For refolding, denatured luciferase was diluted to 50 nm in the same buffer without denaturant and then incubated at 25 °C. The reaction was supplemented with ATP (5 mm), an ATP regenerating system (10 mm phosphocreatine, 100 μg/ml phosphocreatine kinase), 0.15 mg/ml bovine serum albumin, and chaperone proteins as indicated in the figure legends. The luciferase activity was determined in a Beckman scintillation counter using the Luciferase Assay System (Promega E1500). GFP Disaggregation Depends on Both the DnaK and ClpB Chaperones—To study in detail the cooperation between ClpB and the DnaK and co-chaperones in the disaggregation of substrate proteins, we developed a system that allowed us to follow real time measurements of this reaction. We took advantage of the fact that the fluorescence of the GFP depends on its proper folding. Only when the chromophore group is buried inside the β-barrel-like structure can the fluorescence characteristic to GFP be detected (45Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4946) Google Scholar). We destabilized the otherwise extremely stable GFP protein structure by thermal denaturation at 85 °C. Such a treatment resulted in the complete loss of the characteristic fluorescence of GFP (Fig. 1A) and its extensive aggregation. Sizing chromatography (Fig. 1B) revealed that the majority of heat-treated GFP was eluted from the column in the fractions 30–34 corresponding to the molecular mass of ∼1–2 × 106 Da. Similar results were obtained by sedimentation analysis. The majority of heat-treated GFP sedimented in glycerol gradient 2–3-fold faster than the purified RNA polymerase (∼ 400 kDa) (result not shown). Monomers of GFP were not detected either by sizing chromatography or sedimentation analysis (Fig. 1B and result not shown). The GFP aggregates obtained following thermal inactivation did not refold spontaneously, because the characteristic fluorescence was not observed, even following prolonged incubation (Fig. 2A). To measure GFP disaggregation and refolding in real time, protein aggregates were placed in a spectrofluorometric cuvette, ClpB and DnaK/DnaJ/GrpE chaperones were added, and the fluorescence characteristic to GFP was monitored. Following an initial ∼2-min lag phase, GFP fluorescence increased linearly up to 30 min (Fig. 2B). Then the rate of the fluorescence increase became slower. After 100 min of GFP disaggregation and reactivation, the recovered GFP fluorescence reached approximately 30–40% of the fluorescence of nondenatured GFP (Fig. 2). The observed increase in GFP fluorescence correlated well with the formation of GFP monomers, as monitored by sedimentation analysis (result not shown). Thus, we conclude that GFP aggregates are efficiently disaggregated by this bi-chaperone system. In control experiments it was shown that a separate addition of ClpB alone to GFP aggregates did not lead to a fluorescence increase, whereas the addition of DnaK/DnaJ/GrpE alone resulted only in a slight increase in GFP fluorescence (Fig. 2A). This increase of fluorescence most likely reflects the DnaK/DnaJ/GrpE-dependent reactivation of small GFP aggregates (∼100 kDa), little amounts of which were detected in aggregated GFP preparation by sizing chromatography. It is important to mention here that the folding of completely unfolded GFP monomers takes place in 20–30 s and does not require the presence of chaperones (45Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4946) Google Scholar, 46Makino Y. Amada K. Taguchi H. Yoshida M. J. Biol. Chem. 1997; 272: 12468-12474Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Therefore, we assumed that the limiting step of the observed GFP fluorescence increase was not the spontaneous refolding of GFP monomers but the chaperone-dependent disaggregation reaction. The dependence of GFP disaggregation on the presence of chaperones from the Hsp100 and Hsp70 systems enabled us to perform a detailed analysis of the initial steps during the disaggregation reaction.Fig. 2Thermally denatured GFP is reactivated by the ClpB-DnaK/DnaJ/GrpE bi-chaperone system. Increase of the fluorescence of denatured GFP following incubation with ClpB, DnaK, DnaJ, and GrpE; ClpB alone; DnaK, DnaJ, and GrpE; or no chaperones. GFP fluorescence was monitored in the long (A) and short (B) time period at 510 nm following excitation at 395 nm.View Large Image Figure ViewerDownload (PPT) Preincubation of Aggregated GFP with DnaK/DnaJ Results in Faster Start of Disaggregation—To investigate the order of chaperone action in the disaggregation reaction, GFP aggregates were preincubated first in the spectrofluorometric cuvette for 5 min in the presence of ATP with either ClpB or DnaK/DnaJ/GrpE chaperones. Then, at time 0, the missing chaperone components were added, and GFP fluorescence was monitored. When GFP aggregates were first incubated with ClpB, before the addition of the DnaK/DnaJ/GrpE chaperones, the lag phase in the increase of fluorescence was the same as that observed when the complete set of chaperone proteins was added simultaneously (Fig. 3, A and B). In contrast, when GFP aggregates were incubated first with the DnaK/DnaJ/GrpE chaperones, followed by ClpB addition, no lag phase was observed, and GFP fluorescence increased linearly from the moment of ClpB addition (Fig. 3, A and B). In both cases, the efficiency of GFP disaggregation was similar (approximately 40% of the fluorescence of nondenatured GFP). We calculated and compared the rates of GFP refolding during the disaggregation reactions. Based on the shape of the fluorescence curve, we chose two time periods in which the reaction rates were calculated, the initial rate (the average rate in the first 2 min of the reaction) and final rate (the rate during the linear increase of fluorescence measured between 10 and 12 min of the reaction). The initial rate of GFP refolding was ∼4.6-fold higher in the case when aggregated GFP was preincubated with DnaK/DnaJ/GrpE as compared with ClpB preincubation (Fig. 3C). The comparison of the final rates gave nearly the same value regardless of the order of chaperone addition (62.6 ng of GFP refolded per min for DnaK/DnaJ/GrpE preincubation and 59 ng of GFP/min for ClpB preincubation) (Fig. 3C). These results taken together show that the DnaK chaperone system must interact with aggregated GFP prior to ClpB chaperone. To further determine which component of the DnaK system is needed at the preincubation step, GFP aggregates were incubated either with DnaK or DnaJ alone, with both DnaK and DnaJ, or with the whole DnaK system (DnaK/DnaJ/GrpE). Following this, the missing chaperones were added, and GFP fluorescence was monitored. Preincubation of aggregated GFP with DnaK or DnaJ resulted in initial rates comparable with those measured when no chaperones were present at the preincubation step (Fig. 4A). In contrast, preincubation of GFP aggregates with both DnaK and DnaJ resulted in a fast initial rate of fluorescence increase, comparable with the rate measured following preincubation with the entire DnaK/DnaJ/GrpE chaperone system (Fig. 4A). These experiments demonstrate that the simultaneous presence of at least DnaK and DnaJ is required at the initial step of the disaggregation reaction. Presence of ATP Is Required at the Initial Step of Disaggregation—It is known that the affinity of DnaK for substrates depends on the nature of its bound nucleotide and that this process is regulated by the DnaJ co-chaperone. DnaJ also targets various substrates for DnaK binding (26Wawrzynow A. Zylicz M. J. Biol. Chem. 1995; 270: 19300-19306Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Liberek K. Wall D. Georgopoulos C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6224-6228Crossref PubMed Scopus (93) Google Scholar, 28Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (472) Google Scholar, 29Han W. Christen P. J. Biol. Chem. 2003; 278: 19038-19043Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). DnaK and DnaJ were incubated in the spectrofluorometric cuvette with aggregated GFP in the presence of 1 mm ATP, ADP, or ATPγS. After 5 min ClpB an" @default.
• W2110829900 created "2016-06-24" @default.
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• W2110829900 date "2004-10-01" @default.
• W2110829900 modified "2023-10-18" @default.
• W2110829900 title "Successive and Synergistic Action of the Hsp70 and Hsp100 Chaperones in Protein Disaggregation" @default.
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