The Immunological Dissection of the Escherichia Coli Molecular Chaperone Dnaj

FEMS Microbiology Letters 196 (2001) 19^23 www.fems-microbiology.org

The immunological dissection of the Escherichia coli molecular chaperone DnaJ

Salma Al-Herran, William Ashraf *

Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK Downloaded from https://academic.oup.com/femsle/article/196/1/19/474216 by guest on 30 September 2021

Received 6 September 2000; received in revised form 3 November 2000; accepted 8 January 2001

Abstract

In Escherichia coli, one of the main molecular chaperones is DnaJ (hsp40), which mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes. DnaJ is characterised by the presence of four distinct domains: the J-domain, glycine/phenylalanine-rich (G/F), cysteine-rich (Zn-finger) and C-terminal regions. Truncated DnaJ polypeptides (DnaJ 1^108, DnaJ v1^108, DnaJ v1^199) representing these domains were over-produced and used as a source of immunogens for the generation of sequence-specific polyclonal antibodies. Epitope mapping was achieved by Western blotting, which demonstrated the presence of antibodies directed against these domains. These characterised affinity-purified antibodies were then used to assess the role of DnaJ in the protection of firefly luciferase from irreversible heat-inactivation. In this study we have demonstrated the involvement of J-, G/F and Zn-finger domains in the protection of luciferase from heat-inactivation. The C-terminal region had only partial involvement in luciferase protection. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: DnaJ; Escherichia coli; Luciferase

1. Introduction The hsp40 protein family, including DnaJ, are characterised by the combination of four distinct domains: the In Escherichia coli the main heat shock proteins, DnaK J-domain, glycine/phenylalanine-rich (G/F), cysteine-rich (hsp70) and DnaJ (hsp40), are highly conserved molecular (Zn-¢nger) and C-terminal regions (Fig. 1). All hsp40 pro- chaperones and together with GrpE (hsp20) form the teins contain the highly conserved J-domain, correspond- `DnaK chaperone machine' [1]. This machine mediates in ing to a 70-amino acid residue present at the amino-termi- a variety of highly conserved cellular processes including nal portion of the E. coli DnaJ protein [6]. The J-domain is protein-folding reactions, the assembly/disassembly of pro- separated by a 30^40-amino acid glycine/phenylalanine- tein complexes and the protection of proteins from ther- rich (over 40% glycine and 15% phenylalanine) G/F region mal denaturation. which is thought to act as a £exible spacer that separates DnaJ is a dimeric chaperone, which binds to nascent the J-domain from other parts of the protein [7]. A recent polypeptides emerging from ribosomes aiding correct pro- study, involving spectroscopic analysis of 5-hydroxy- tein folding, and in addition preventing the aggregation of tryptophan-labelled proteins, has demonstrated that the denatured proteins. DnaK binds to DnaJ-substrate form- J-domain is a single well-ordered domain, whilst the G/F ing a stable ternary complex. DnaJ, possibly via the N- region shows a lack of well-ordered structure [8]. The terminal QKRAA sequence [2], then stimulates the weak third additional conserved domain is the cysteine-rich re- ATPase activity of DnaK and on addition of GrpE the gion that corresponds to a 150-amino acid stretch at the substrate is released. DnaJ will also bind to folded poly- centre of the protein that has the four repeated sequence peptides, such as c32 and bacteriophage RepA protein (for motifs, CXXCXGXG, where X denotes a charged or po- reviews see [3^5]). lar amino acid residue. It has been shown that each mol- ecule of E. coli DnaJ binds two zinc ions that are tetrahe- drally co-ordinated by the sulfur atoms of four cysteine * Corresponding author. Tel: +44 (1274) 233589; residues. The cysteine-rich domain, is in fact two Zn ¢n- Fax: +44 (1274) 309742; E-mail: [email protected] gers that may form a pocket with a hydrophobic core. The

FEMSLE 9797 8-3-01 20 S. Al-Herran, W. Ashraf / FEMS Microbiology Letters 196 (2001) 19^23 solution structure of this region has been elucidated by (w/v) sucrose. The suspension was then frozen in liquid nuclear magnetic resonance and contains a novel V-shaped nitrogen and stored at 380³C until required. fold with an extended L-hairpin topology [9]. The C-terminal, representing approximately one third of DnaJ, does 2.2. Puri¢cation of DnaJ and truncated proteins not contain any consensus motifs and this region is the least conserved and believed to be involved in substrate Protein puri¢cation required a three-step process involv- binding [10,11]. ing Q-Sepharose0 HP (Pharmacia Amersham Biotech), The DnaK/DnaJ/GrpE chaperone machine suppresses hydroxyapatite (Bio-Rad) and P11-cellulose phosphate aggregation of both prokaryotic and eukaryotic polypep- (Whatman) column chromatography. The method was as tides, thus promoting protein folding (for review see [12]). previously described in [17] except that the ammonium This chaperone machine can also protect partially aggre- sulfate precipitation step was omitted. gated enzymes, such as E. coli RNA polymerase, DNA

polymerase II and ¢re£y luciferase. DnaJ alone can also 2.3. Generation and puri¢cation of anti-DnaJ antibodies Downloaded from https://academic.oup.com/femsle/article/196/1/19/474216 by guest on 30 September 2021 protect luciferase and rhodanase from thermal denaturation [13,14]. Antisera were raised in female New Zealand white rab- In this study, characterised sequence-speci¢c polyclonal bits by administration of 100 Wg puri¢ed DnaJ protein and anti-DnaJ antibodies (Fig. 1) were employed as inhibitors truncated proteins. The procedure was repeated at regular to identify which domains on DnaJ are responsible for its intervals over a 6-week period. Antibodies were puri¢ed ability to protect and reactivate heat-inactivated luciferase. using a Sepharose^DnaJ a¤nity column generated from Previous studies have used randomly generated monoclo- 1 ml Hi Trap0 NHS-activated a¤nity column according nal antibodies in order to gain functional understanding of to the manufacturer's instructions (Pharmacia Amersham E. coli c32 [15] and DnaK [16]. However, our immunolog- Biotech). A¤nity-puri¢ed antibodies were evaluated by ical dissection has been programmed to ensure that anti- Western blotting and then stored at 320³C until required. bodies are directed against all DnaJ domains. 2.4. Epitope mapping

2. Materials and methods Western blotting was used to epitope map a¤nity-puri- ¢ed sequence-speci¢c antibodies directed against DnaJ 1^ 2.1. E. coli strains, plasmids and growth conditions 108, DnaJ v1^108 and DnaJ v1^199 and also polyclonal antibodies against full-length DnaJ. SDS^PAGE analysis E. coli strains and plasmids used in this study are listed of proteins was carried out using 12.5% (w/v) polyacryl- in Table 1 [11,17]. Cells were grown aerobically at 37³C in amide gels, which were then electroblotted onto nitrocel- Luria broth (LB) supplemented with 100 Wgml31 ampi- lulose. Immunological detection of DnaJ polypeptides was cillin. Full-length DnaJ was over-produced from E. coli achieved using anti-DnaJ antibodies and a horseradish B178 pMOB45-dnaJ+12 grown in LB supplemented with peroxidase conjugate anti-rabbit IgG (Dako) system [18]. 30 Wgml31 chloramphenicol. Truncated DnaJ polypeptides were over-produced using plasmids pDW19-dnaJ12, 2.5. Protection of luciferase from irreversible pAED4-dnaJv1^108 and pAED4-dnaJv1^199 (derivatives heat-inactivation of the pET vector) after CaCl2 transformation into com- petent E. coli BL21 (DE3). Expression of dnaJ was in- Fire£y luciferase (Sigma) was stored at 5 Wgml31 in 1 M duced at OD600 0.1 by the addition of 50 WM isopropyl (pH 7.4) glycylglycine and kept in aliquots at 320³C. The L-D-thiogalactopyranoside. Cultures were harvested at assay was adapted from a previously published method

OD600 0.8 by centrifugation (7000Ug for 10 min at 4³C) [19]. Luciferase (250 ng) was inactivated by incubation and then resuspended in 50 mM Tris^HCl (pH 8.0), 10% at 42³C for 10 min in the presence of 1 Wg DnaK, 600 ng DnaJ and 400 ng GrpE. The reactivation mixture (50 Wl)

Table 1 Bacterial strains used in this study Strain Genotype Plasmid Source/reference E. coli B178 araD139, v(argF-lac) U169, rpsL 150, relA1, pMOB45-dnaJ [17] deoC1, ptsF25, rpsR, £bB301 3 E. coli BL21 (DE3) F dcm, omp,T hsd (rB mB), gal V (DE3) none Stratagene E. coli BL21(DE3)-DnaJ v1^108 as above pAED4-dnaJv1^108 [11] E. coli BL21(DE3)-DnaJ v1^199 as above pAED4-dnaJv1^199 [11] E. coli BL21(DE3) as above pDW19-dnaJ12 [11]

FEMSLE 9797 8-3-01 S. Al-Herran, W. Ashraf / FEMS Microbiology Letters 196 (2001) 19^23 21 Downloaded from https://academic.oup.com/femsle/article/196/1/19/474216 by guest on 30 September 2021

Fig. 1. Structural features of E. coli DnaJ and epitope mapping summary. Adapted from [20]. contained bu¡er A (20 mM Tris^HCl (pH 7.5), 100 mM 3. Results and discussion

KCl, 40 mM NaCl, 10 mM MgCl2, 0.05 mM EDTA, 5 mM DTT). The protection assay was initiated by the To investigate the functional roles of the conserved addition of 4 mM ATP and then 1 Wl of each reaction DnaJ domains, a series of a¤nity-puri¢ed sequence-specif- was diluted into 60 Wl of luciferase assay mixture (50 Wl ic antibodies were used to provide an alternative, sensitive of luciferase assay substrate solution (Promega) and 10 Wl and speci¢c means of understanding the function. These of lysis bu¡er reagent (Promega) containing 6 mg ml31 polyclonal antibodies were used as inhibitors to assess the bovine serum albumin (BSA; Sigma)). Activity was mea- role of de¢ned DnaJ domains in the protection of lucifer- sured using a 1250 Luminometer (LKB-Wallac). Relative ase from heat-inactivation. A summary of the epitope luciferase activity was calculated by taking as 100% the mapping results is detailed in Fig. 1. For the sake of brev- activity exhibited by control non-heat-treated luciferase. ity the Western blotting data has not been presented. The results presented are the means of at least three in- Western blot analysis con¢rmed the production of anti- dependent experiments. bodies against full-length DnaJ and DnaJ polypeptide se- Puri¢ed anti-DnaJ, anti-DnaJ 1^108, anti-DnaJ v1^108 quences. Anti-DnaJ v1^108 antibodies only produced a and anti-DnaJ v1^199 were used as inhibitors of the lu- weak signal against DnaJ v1^199 protein and failed to ciferase protection. The procedure was carried out as de- recognise the DnaJ 1^108 (J-domain and G/F domain). tailed above except that DnaJ was pre-incubated in the This result strongly supports the notion that antibodies presence of antibody molecules (1:10 molar ratio) for 90 raised against DnaJ v1^108 were more speci¢c for the min at 24³C prior to the heat-inactivation. Puri¢ed pre- 109^199-amino acid sequence (90 amino acids) which con- immune sera were also included as a control. tains the Zn-¢nger-like domain. From these results we speculate that the Zn-¢nger-like domain is potentially im- 2.6. Protein determination munodominant. Anti-DnaJ v1^199 recognised all DnaJ polypeptides except for DnaJ 1^108. Anti-DnaJ 1^108 Protein quanti¢cation was performed using the Brad- ford reagent (0.01% w/v Coomassie brilliant blue, 8.5% Table 2 v/v phosphoric acid and 5% v/v methanol) supplied by Epitope mapping results for anti-DnaJ sequence-speci¢c antibodies Bio-Rad and used according to the manufacturer's in- Sequence-speci¢c anti- Maximum number of Domains structions. Samples were measured for protein using an body amino acids recognised Ultrospec II (LKB, Biochrom) spectrophotometer at 595 Anti-DnaJ 1^108 108 J- and G/F nm. Protein concentration was calculated from a standard Anti-DnaJ v1^108 90 Zn-¢ngers curve using BSA as a standard. Anti-DnaJ v1^199 176 C-terminal region

FEMSLE 9797 8-3-01 22 S. Al-Herran, W. Ashraf / FEMS Microbiology Letters 196 (2001) 19^23

the scale used). This result was examined in more detail through the application of characterised anti-DnaJ sequence antibodies (see Fig. 1). Anti-DnaJ 1^108, anti- DnaJ v1^108 and anti-DnaJ v1^199 sequence-speci¢c antibodies are directed against the highly conserved J-domain, Zn-¢nger-like domain and putative substrate-binding domain, respectively. Antibodies against DnaJ 1^108 almost completely inhibited the reactivation of denatured luciferase identifying this region as necessary for function- ing of the Hsp70 chaperone machine. This supports results from a previous study, which demonstrated the role of the J-domain in the stimulation of the ATPase activity DnaK

[11]. However, a more recent study has shown that the Downloaded from https://academic.oup.com/femsle/article/196/1/19/474216 by guest on 30 September 2021 J-domain by itself neither stimulates ATPase activity of DnaK nor supports the replication of bacteriophage V Fig. 2. Antibody inhibition of the protection of luciferase from irrevers- DNA [21]. Antibodies against DnaJ v1^108, which were ible heat-inactivation. Control reactivation reaction mixture (50 Wl) con- only directed against the Zn-¢nger domains, blocked the taining bu¡er A (20 mM Tris^HCl (pH 7.5), 40 mM NaCl, 10 mM function of this domain in our protection assay. This re- MgCl2, 0.05 mM EDTA, 5 mM DTT) with 250 ng of ¢re£y luciferase (Sigma) with or without 600 ng of DnaJ, 1 Wg of DnaK, and 400 ng of sult is in good agreement with a study which demonstrated GrpE (F). After inactivation at 42³C for 10 min, the reaction was incu- that hdj2 (homologous to DnaJ), but not hdj1 (lacking the bated at 24³C with 4 mM ATP. At the times indicated, 1-Wl aliquots Zn-¢nger domains), together with hsc70 refolded luciferase were withdrawn, diluted 1:50 and analysed for luciferase activity as de- e¤ciently [22]. scribed in Section 2. Inhibition experiments were conducted as above, except that the DnaJ was pre-incubated in the presence of antibody In a previous study using a DnaK monoclonal antibody molecules (DnaJ:antibody, 1:10 molar ratio) for 90 min at 24³C prior only 25% ATPase inhibition was observed at a stoichiom- to the heat-inactivation of luciferase. Anti-DnaJ (a), anti-DnaJ 1^108 etry of 1:10 [16]. Therefore, we propose that antibodies (P) anti-DnaJ v1^108 (S), anti-DnaJ v1^199 (b). The results presented directed against the DnaJ N-terminal domains are power- are the means of at least three independent experiments. Please note ful inhibitors (from Fig. 2 almost 100% inhibition was that the data points for anti-DnaJ and anti-DnaJ v1^108 partially obscure each other on the scale used. achieved at a stoichiometry of 1:10) of Hsp70 chaperone machine function. The most probable mechanism will in- volve inhibition of DnaJ-mediated stimulation of DnaK only recognised DnaJ 1^108 and full-length DnaJ. The ATPase activity [23]. Whilst the possibility of steric hin- maximum number of amino acids and domains recognised drance and/or e¡ects cannot be entirely discounted we by a¤nity-puri¢ed anti-DnaJ sequence-speci¢c antibodies believe this unlikely as only one antibody, anti-DnaJ 1^ is detailed in Table 2. Antibody titration assays (data not 108, inhibits DnaJ^DnaK complex formation as deter- shown) against full-length DnaJ showed that in all sam- mined in enzyme-linked immunosorbent assays. For exam- ples, each antibody was sensitive at s 1037 dilution. ple, the binding of anti-DnaJ v1^108, as demonstrated by Luciferase was used as a model for functional studies 100% inhibition of luciferase refolding does not sterically because (i) its refolding does not require GroEL/GroES hinder DnaJ^DnaK interaction (data not shown). chaperones and (ii) the luminescence-based enzyme assay Antibodies against the C-terminal domain (anti-DnaJ is extremely sensitive, thus allowing for refolding studies in v1^199) did not have a dramatic inhibitory e¡ect on the small volumes. The in£uence of the Hsp70 chaperone ma- protection of luciferase compared with the other antibod- chine on the thermal protection of luciferase function is ies tested. The results show that in the presence of C-ter- shown in Fig. 2. Protection from heat-inactivation was minal antibodies there is a delay in reactivation, only only demonstrated when all components of the DnaK achieving 80% reactivation after 45 min, in contrast with chaperone machine were present with luciferase, as previ- the control, which achieved 80% after 30 min. Previous ously reported in [13]. A¤nity-puri¢ed antibodies were studies have reported that only full-length DnaJ can co- then used to investigate the involvement of de¢ned DnaJ operate with DnaK and GrpE in refolding denatured lu- domains, the J-domain and the G/F region, Zn-¢nger-like ciferase [14]. Our results are somewhat at variance with and substrate-binding domains, in the protection of lucif- this hypothesis and may be due to the fact that our anti- erase from heat-inactivation. Control experiments with af- bodies did not block all of the available epitopes contained ¢nity-puri¢ed antibodies from pre-immune sera did not within the C-terminus, some of which maybe necessary for inhibit the luciferase assay. chaperone machine function. Fig. 2 shows almost complete inhibition of the reactiva- In summary, our functional studies, using characterised tion of heat-inactivated luciferase with full-length anti- sequence-speci¢c antibodies, demonstrate pivotal roles for DnaJ (please note that the data points for anti-DnaJ the J, G/F and Zn-¢nger-like domains of DnaJ in the and anti-DnaJ v1^108 partially obscure each other on protection of luciferase from thermal denaturation. Pre-

FEMSLE 9797 8-3-01 S. Al-Herran, W. Ashraf / FEMS Microbiology Letters 196 (2001) 19^23 23 vious work on DnaJ has mainly concentrated on the conserved J-domain. Proc. Natl. Acad. Sci. USA 91, 11343^ J-domain [7,11,24] with the Zn-¢nger domain receiving 11347. [8] Greene, M.K., Steede, N.K. and Landry, S.J. (2000) Domain-speci¢c less attention [14], however, this work also supports the spectroscopy of 5-hydroxytryptophan-containing variants of Escheri- view for a signi¢cant role for the latter in molecular chap- chia coli DnaJ. Biochim. Biophys. Acta 1480, 267^277. erone function. Recent studies in E. coli have demonstrated [9] Martinez-Yamout, M., Legge, G.B., Zhang, O., Wright, P.E. and the presence of a novel Hsc66^Hsc20 (J-type accessory pro- Dyson, H.J. (2000) Solution structure of the cysteine-rich domain tein) chaperone system which has been shown to refold of the Escherichia coli chaperone protein DnaJ. J. Mol. Biol. 300, 805^818. denatured luciferase [25]. Therefore, it would be interesting [10] Cyr, D.M. (1997) Hsp40 (DnaJ-related) proteins: an overview. In: to conduct a complementary immunological dissection of Guidebook to Molecular Chaperones and Protein-folding Catalysts Hsc20 using the antibodies generated in this study. (Gething, M.J., Ed.). Oxford University Press, Oxford. [11] Wall, D., Zylicz, M. and Georgopoulos, C. (1994) Identi¢cation of the conserved domain of the Escherichia coli DnaJ protein that stimulates DnaK's ATPase activity and required for V DNA replication.

Acknowledgements Downloaded from https://academic.oup.com/femsle/article/196/1/19/474216 by guest on 30 September 2021 J. Biol. Chem. 269, 5446^5451. [12] Hendrick, J.P. and Hartl, F.U. (1993) Molecular chaperone functions We wish to thank Dr. D. Wall (Stanford, USA) for the of heat shock proteins. Annu. Rev. Biochem. 162, 349^384. generous gifts of DnaJ over-producing strains. W.A. is [13] Schroder, H., Langer, T., Hartl, F.U. and Bukau, B. (1993) DnaK, grateful for grants received from the Nu¤eld Foundation DnaJ, GrpE form a cellular machine capable of repairing heat-in- duced protein damage. EMBO J. 12, 4137^4144. and Society for General Microbiology. S.A.H. is also [14] Szabo, A., Korrszun, R., Hartl, F.U. and Flanagan, J. (1996) A zinc grateful to the British Council (Kuwait) for ¢nancial as- ¢nger-like domain of the molecular chaperone DnaJ is involved in sistance. We are also grateful to Mr. P. Thompkins (Uni- binding to denatured protein substrates. EMBO J. 15, 408^417. versity of Bradford) for critically reviewing this manu- [15] Lesley, S.A., Thompson, N.E. and Burgess, R.R. (1987) Studies of script. the role of the Escherichia coli heat shock regulatory protein c32 by the use of monoclonal antibodies. J. Biol. Chem. 262, 5404^5407. [16] Krska, J., Elthon, T. and Blum, P. (1993) Monoclonal antibody recognition and function of a DnaK (Hsp70) epitope found in References Gram-negative bacteria. J. Bacteriol. 175, 6433^6440. [17] Zylicz, M., Yamamoto, T., McKiittrick, N., Snell, S. and Georgo- [1] Georgopoulos, C. (1992) The emergence of the chaperone machines. poulos, C. (1985) Puri¢cation and properties of the DnaJ replication Trends Biochem. Sci. 17, 295^299. protein of Escherichia coli. J. Biol. Chem. 260, 7591^7598. [2] Auger, I. and Roudier, J. (1997) A function for the QKRAA amino [18] Harlow, E. and Lane, D. (1988) Antibodies: a Laboratory Manual. acid motif mediating binding of DnaJ to DnaK (implications for the Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. association of rheumatoid arthritis with HLA-DR4). J. Clin. Invest. [19] Wickner, S., Gottesman, S., Skowyra, D., Hoskins, J., McKenney, K. 99, 1818^1822. and Maurizi, M.R. (1994) A molecular chaperone, ClpA, functions [3] Morimoto, R., Tissieres, A. and Georgopoulos, C. (Eds.) (1994) The like DnaK and DnaJ. Proc. Natl. Acad. Sci. USA 91, 12218^12222. Biology of Heat Shock Proteins and Molecular Chaperones. Cold [20] Al-Herran, S. and Ashraf, W. (1998) Physiological consequences of Spring Harbor Laboratory, Cold Spring Harbor, NY. the over-production of Escherichia coli truncated molecular chaper- [4] Georgopoulos, C., Liberek, K., Zylicz, M. and Ang, D. (1994) Prop- one DnaJ. FEMS Microbiol. Lett. 162, 117^122. erties of the heat shock proteins of Escherichia coli and the autore- [21] Karzai, A.W. and McMachen, R. (1996) A bipartite signalling mech- gulation of the heat shock response. In: The Biology of Heat Shock anism involved in DnaJ-mediated activation of the Escherichia coli Proteins and Molecular Chaperones (Morimoto, R., Tissieres, A. and DnaK protein. J. Biol. Chem. 271, 11236^11246. Georgopoulos, C., Eds.), pp. 209^249. Cold Spring Harbor Labora- [22] Terada, K., Kanazawa, M., Bukau, B. and Mori, M. (1997) The tory, Cold Spring Harbor, NY. human DnaJ homologue dnaJ2 facilitates mitochondrial protein im- [5] Cheetham, M.E. and Caplan, A.J. (1998) Structure, function and port and luciferase refolding. J. Cell Biol. 139, 1089^1095. evolution of DnaJ: conservation and adaptation of chaperone func- [23] Mayer, P.M., Schroder, H., Rudiger, S., Paal, K., Laufer, T. and tion. Cell Stress Chaperone 3, 28^36. Bukau, B. (2000) Mutlistep mechanism of substrate binding deter- [6] Georgopoulos, C. and Welch, W.J. (1993) Role of the major heat mines chaperone activity of Hsp70. Nat. Struct. Biol. 7, 587^593. shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9, [24] Huang, K., Flanagan, J.M. and Prestegard, J.H. (1999) The in£uence 601^634. of C-terminal extension on the structure of the `J-domain' in E. coli [7] Szyperski, T., Pellecchia, M., Wall, D., Georgopoulos, C. and Wuth- DnaJ. Protein Sci. 8, 203^214. rich, K. (1994) NMR structure determination of the Escherichia coli [25] Silberg, J.J., Ho¡, K.G. and Vickery, L.E. (1998) The Hsc66^Hsc20b DnaJ molecular chaperone: secondary structure and backbone chaperone system in Escherichia coli: chaperone activity and interac- fold of the N-terminal region (residues 2^108) containing the highly tions with DnaK^DnaJ^GrpE system. J. Bacteriol. 180, 6617^6624.

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