Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK.


Abstract

The thermal unfolding of the Escherichia coli 70 kDa heat shock protein, DnaK, exhibits three well defined transitions. At pH 7.6, these transitions are centered at 45.2, 58.0 and 73.3 degrees C. High sensitivity calorimetric scans as a function of pH indicate that the folding/unfolding behavior is well described by a four-state model which includes a delta H, tm and delta Cp for each state. Calorimetric scans of a 44 kDa N-terminal proteolytic fragment show a major transition centered at 47.5 degrees C (N1) and a minor transition at 79.4 degrees C (N2). A calorimetric scan of a 23 kDa C-terminal proteolytic fragment exhibits a low temperature peak at 58.5 degrees C (C1) and a high temperature peak at 70.6 degrees C (C2). Deconvolution analysis of the low temperature peak reveals that it is actually composed of two transitions of roughly equal delta H centered at 50.4 degrees C (C1a) and 58.2 degrees C(C1b). These experiments have allowed us to assign the transitions of the intact protein as follows. The low temperature transition of DnaK can be assigned to the N-terminal region on the basis of the similarity between the delta H and tm values for the low temperature transition and those obtained for the N1 transition of the isolated N-terminal fragment. This assignment is also supported by measurements of the intrinsic fluorescence emission as a function of temperature. DnaK contains a single tryptophan localized at residue 102 in the N-terminal domain of the protein. Additionally, calorimetric scans show that the tm of the low temperature transition increases by 9.2 degrees C in the presence of excess ADP, which is known to bind to the N-terminal domain. The middle transition can be assigned to the C1a and C1b transitions of the C-terminal fragment on the basis of the similarity of delta H and tm. In the intact protein C1a and C1b form a single cooperative unit; however, the cooperative interactions between these folding/unfolding domains are disrupted in the isolated fragment. The high temperature transition of the intact protein is composed of contributions from both the N-terminal and C-terminal regions of the protein. These studies have allowed us to develop a quantitative model of the folding/unfolding behavior of DnaK. Study holds ProTherm entries: 9246, 9247, 9248, 9249, 9250, 9251, 9252, 9253, 9254, 9255, 9256, 9257, 9258, 9259, 9260, 9261, 9262, 9263 Extra Details: Transition (1) heat shock proteins; protein folding; folding intermediates;,protein thermodynamics; molecular chaperones

Submission Details

ID: XFiFYfWd

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Montgomery D;Jordan R;McMacken R;Freire E,J. Mol. Biol. (1993) Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK. PMID:8102181
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1Q5L 2003-11-04 NMR structure of the substrate binding domain of DnaK bound to the peptide NRLLLTG
1BPR 1999-03-02 NMR STRUCTURE OF THE SUBSTRATE BINDING DOMAIN OF DNAK, MINIMIZED AVERAGE STRUCTURE
1DG4 1999-12-08 NMR STRUCTURE OF THE SUBSTRATE BINDING DOMAIN OF DNAK IN THE APO FORM
2BPR 1999-03-02 NMR STRUCTURE OF THE SUBSTRATE BINDING DOMAIN OF DNAK, 25 STRUCTURES
2KHO 2009-05-12 NMR-RDC / XRAY structure of E. coli HSP70 (DNAK) chaperone (1-605) complexed with ADP and substrate
4F01 2013-04-17 1.4 Crystal structure of an artificial dimeric DnaK complex
4HY9 2013-04-17 1.55 Crystal structure of the substrate binding domain of E.coli DnaK in complex with pyrrhocoricin_LYZZ (residues 1 to 11)
4JNF 2013-05-29 1.62 Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP
4EZP 2013-04-17 1.65 Crystal structure of the substrate binding domain of E.coli DnaK in complex with A3-APO(residues 1 to 20)
4HYB 2013-04-17 1.7 Crystal structure of the substrate binding domain of E.coli DnaK in complex with pyrrhocoricin_LYZI (residues 1 to 10)
4EZX 2013-04-17 1.7 Crystal structure of the substrate binding domain of E.coli DnaK in complex with the designer peptide NRLMLTG
4R5K 2014-09-10 1.75 Crystal structure of the DnaK C-terminus (Dnak-SBD-B)
4EZW 2013-04-17 1.8 Crystal structure of the substrate binding domain of E.coli DnaK in complex with the designer peptide NRLLLTG
4JWC 2013-11-13 1.8 Crystal structure of the substrate binding domain of E.coli DnaK in complex with bovine Bac7(1-16)
4EZN 2013-04-17 1.8 Crystal structure of the substrate binding domain of E.coli DnaK in complex with pyrrhocoricin
4EZY 2013-04-17 1.85 Crystal structure of the substrate binding domain of E.coli DnaK in complex with the designer peptide NRLILTG
4EZS 2013-04-24 1.9 Crystal structure of the substrate binding domain of E.coli DnaK in complex with metchnikowin (residues 20 to 26)
4EZU 2013-04-24 1.9 Crystal structure of the substrate binding domain of E.coli DnaK in complex with PR-bombesin in space group I222
4EZR 2013-04-17 1.9 Crystal structure of the substrate binding domain of E.coli DnaK in complex with the C-terminal part of drosocin (residues 12 to 19)
4EZO 2013-04-17 1.9 Crystal structure of the substrate binding domain of E.coli DnaK in complex with PR-39 (residues 1 to 15)
4JWI 2013-11-13 1.9 Crystal structure of the substrate binding domain of E.coli DnaK in complex with sheep Bac7(35-43)
4E81 2012-05-30 1.9 Crystal structure of the substrate binding domain of E.coli DnaK in complex with a short apidaecin peptide
4JWE 2013-11-13 1.95 Crystal structure of the substrate binding domain of E.coli DnaK in complex with sheep Bac7(1-21)
4F00 2013-04-17 1.95 Crystal structure of the substrate binding domain of E.coli DnaK in complex with an apidaecin fragment from the bumblebee (residues 3 to 11)
4JWD 2013-11-13 1.95 Crystal structure of the substrate binding domain of E.coli DnaK in complex with bovine Bac7(15-28)
4JNE 2013-05-29 1.96 Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP
4R5I 2014-09-10 1.97 Crystal structure of the DnaK C-terminus with the substrate peptide NRLLLTG
4EZT 2013-04-17 2.0 Crystal structure of the substrate binding domain of E.coli DnaK in complex with heliocin (residues 14 to 21)
1DKZ 1996-12-07 2.0 THE SUBSTRATE BINDING DOMAIN OF DNAK IN COMPLEX WITH A SUBSTRATE PEPTIDE, DETERMINED FROM TYPE 1 NATIVE CRYSTALS
4EZQ 2013-04-17 2.0 Crystal structure of the substrate binding domain of E.coli DnaK in complex with the C-terminal part of pyrrhocoricin (residues 12 to 20)
1DKX 1996-12-07 2.0 THE SUBSTRATE BINDING DOMAIN OF DNAK IN COMPLEX WITH A SUBSTRATE PEPTIDE, DETERMINED FROM TYPE 1 SELENOMETHIONYL CRYSTALS
4EZZ 2013-04-17 2.05 Crystal structure of the substrate binding domain of E.coli DnaK in complex with the designer peptide ELPLVKI
4EZV 2013-04-24 2.1 Crystal structure of the substrate binding domain of E.coli DnaK in complex with PR-bombesin in space group P21212
3DPO 2009-03-10 2.1 Crystal structure of the substrate binding domain of E. coli DnaK in complex with a short pyrrhocoricin-derived inhibitor peptide
3QNJ 2011-03-23 2.28 Crystal structure of the substrate binding domain of E.coli DnaK in complex with the antimicrobial peptide oncocin
4JN4 2013-05-29 2.3 Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP
4R5J 2014-09-10 2.36 Crystal structure of the DnaK C-terminus (Dnak-SBD-A)
4B9Q 2012-11-14 2.4 Open conformation of ATP-bound Hsp70 homolog DnaK
3DPP 2009-03-10 2.5 Crystal structure of the substrate binding domain of E. coli DnaK in complex with a long pyrrhocoricin-derived inhibitor peptide (form A)
3DPQ 2009-03-10 2.6 Crystal structure of the substrate binding domain of E. coli DnaK in complex with a long pyrrhocoricin-derived inhibitor peptide (form B)
1DKG 1997-08-20 2.8 CRYSTAL STRUCTURE OF THE NUCLEOTIDE EXCHANGE FACTOR GRPE BOUND TO THE ATPASE DOMAIN OF THE MOLECULAR CHAPERONE DNAK
1DKY 1996-12-07 2.8 THE SUBSTRATE BINDING DOMAIN OF DNAK IN COMPLEX WITH A SUBSTRATE PEPTIDE, DETERMINED FROM TYPE 2 NATIVE CRYSTALS
5OOW 2018-04-25 2.9 Crystal structure of lobe II from the nucleotide binding domain of DnaK in complex with AMPPCP
4R5L 2014-09-10 2.97 Crystal structure of the DnaK C-terminus (Dnak-SBD-C)
5NRO 2018-01-17 3.25 Structure of full-length DnaK with bound J-domain
4R5G 2014-09-10 3.45 Crystal structure of the DnaK C-terminus with the inhibitor PET-16

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.6 Chaperone protein DnaK A7FME3 DNAK_YERP3
91.1 Chaperone protein DnaK B4F2V5 DNAK_PROMH
91.1 Chaperone protein DnaK B1JL04 DNAK_YERPY
91.1 Chaperone protein DnaK Q66ET0 DNAK_YERPS
91.1 Chaperone protein DnaK A4TQF9 DNAK_YERPP
91.1 Chaperone protein DnaK Q1CMV7 DNAK_YERPN
91.1 Chaperone protein DnaK A9R015 DNAK_YERPG
91.1 Chaperone protein DnaK Q8ZIM7 DNAK_YERPE
91.1 Chaperone protein DnaK B2K3M0 DNAK_YERPB
91.1 Chaperone protein DnaK Q1C0J9 DNAK_YERPA
91.1 Chaperone protein DnaK A1JJD5 DNAK_YERE8
92.2 Chaperone protein DnaK Q6D0B7 DNAK_PECAS
92.8 Chaperone protein DnaK C6DF10 DNAK_PECCP
95.0 Chaperone protein DnaK A4W6D5 DNAK_ENT38
95.1 Chaperone protein DnaK A7MIK5 DNAK_CROS8
95.0 Chaperone protein DnaK B5Y242 DNAK_KLEP3
95.9 Chaperone protein DnaK B5R5I2 DNAK_SALEP
95.1 Chaperone protein DnaK A6T4F4 DNAK_KLEP7
96.2 Chaperone protein DnaK Q56073 DNAK_SALTY
96.2 Chaperone protein DnaK Q8Z9R1 DNAK_SALTI
96.2 Chaperone protein DnaK B4TVZ5 DNAK_SALSV
96.2 Chaperone protein DnaK B5BLH8 DNAK_SALPK
96.2 Chaperone protein DnaK C0Q4F3 DNAK_SALPC
96.2 Chaperone protein DnaK A9MXI2 DNAK_SALPB
96.2 Chaperone protein DnaK Q5PDJ5 DNAK_SALPA
96.2 Chaperone protein DnaK B4T6D6 DNAK_SALNS
96.2 Chaperone protein DnaK B4TIB4 DNAK_SALHS
96.2 Chaperone protein DnaK B5RF08 DNAK_SALG2
96.2 Chaperone protein DnaK B5FHA6 DNAK_SALDC
95.6 Chaperone protein DnaK Q57TP3 DNAK_SALCH
96.2 Chaperone protein DnaK B5F6Y8 DNAK_SALA4
96.1 Chaperone protein DnaK A9MR77 DNAK_SALAR
95.6 Chaperone protein DnaK A8ALU3 DNAK_CITK8
100.0 Chaperone protein DnaK Q3Z601 DNAK_SHISS
99.8 Chaperone protein DnaK Q83MH5 DNAK_SHIFL
99.8 Chaperone protein DnaK Q0T8H6 DNAK_SHIF8
100.0 Chaperone protein DnaK Q32KA5 DNAK_SHIDS
100.0 Chaperone protein DnaK Q326K7 DNAK_SHIBS
100.0 Chaperone protein DnaK Q1RGI8 DNAK_ECOUT
100.0 Chaperone protein DnaK P0A6Y8 DNAK_ECOLI
100.0 Chaperone protein DnaK B1IRG0 DNAK_ECOLC
100.0 Chaperone protein DnaK P0A6Y9 DNAK_ECOL6
100.0 Chaperone protein DnaK Q0TLX5 DNAK_ECOL5
100.0 Chaperone protein DnaK A1A766 DNAK_ECOK1
98.3 Chaperone protein DnaK A7ZVV7 DNAK_ECOHS
100.0 Chaperone protein DnaK P0A6Z0 DNAK_ECO57
100.0 Chaperone protein DnaK A7ZHA4 DNAK_ECO24
90.5 Chaperone protein DnaK A8G9K8 DNAK_SERP5