TY - JOUR
T1 - Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK
AU - Montgomery, Diana
AU - Jordan, Robert
AU - McMacken, Roger
AU - Freire, Ernesto
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1993/1/1
Y1 - 1993/1/1
N2 - 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°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 ΔH, tm and ΔCp for each state. Calorimetric scans of a 44 kDa N-terminal proteolytic fragment show a major transition centered at 47.5°C (N1) and a minor transition at 79.4°C (N2). A calorimetric scan of a 23 kDa C-terminal proteolytic fragment exhibits a low temperature peak at 58.5°C (C1) and a high temperature peak at 70.6°C (C2). Deconvolution analysis of the low temperature peak reveals that it is actually composed of two transitions of roughly equal ΔH centered at 50.4°C (C1a) and 58.2°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 ΔH 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°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 ΔH 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.
AB - 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°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 ΔH, tm and ΔCp for each state. Calorimetric scans of a 44 kDa N-terminal proteolytic fragment show a major transition centered at 47.5°C (N1) and a minor transition at 79.4°C (N2). A calorimetric scan of a 23 kDa C-terminal proteolytic fragment exhibits a low temperature peak at 58.5°C (C1) and a high temperature peak at 70.6°C (C2). Deconvolution analysis of the low temperature peak reveals that it is actually composed of two transitions of roughly equal ΔH centered at 50.4°C (C1a) and 58.2°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 ΔH 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°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 ΔH 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.
KW - Folding intermediates
KW - Heat shock proteins
KW - Molecular chaperones
KW - Protein folding
KW - Protein thermodynamics
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U2 - 10.1006/jmbi.1993.1418
DO - 10.1006/jmbi.1993.1418
M3 - Article
C2 - 8102181
AN - SCOPUS:0027172465
SN - 0022-2836
VL - 232
SP - 680
EP - 692
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -