Thermodynamic and Structural Analysis of the Folding/Unfolding Transitions of the Escherichia coli Molecular Chaperone DnaK

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^oC. High sensitivity calorimetric scans as a function of pH indicate that the folding/unfolding behavior is well de... Ausführliche Beschreibung

1. Person: Montgomery, D.
Weitere Personen: Jordan, R.; McMacken, R.; Freire, E.
Quelle: in Journal of Molecular Biology Vol. 232, No. 2 (1993), p. 680-692
Weitere Artikel
Format: Online-Artikel
Genre: heat shock proteins, protein folding, folding intermediates, protein thermodynamics, molecular chaperones
Sprache: English
Veröffentlicht: 1993
Beschreibung: Online-Ressource
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Anmerkung: Copyright: Copyright (c) 2007 Elsevier Ltd
Zusammenfassung: 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^oC. 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, t"m and ΔC"p for each state. Calorimetric scans of a 44 kDa N-terminal proteolytic fragment show a major transition centered at 47.5^oC (N1) and a minor transition at 79.4^oC (N2). A calorimetric scan of a 23 kDa C-terminal proteolytic fragment exhibits a low temperature peak at 58.5^oC (C1) and a high temperature peak at 70.6^oC (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^oC (Cla) and 58.2^oC (Clb). 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 t"m 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 t"m of the low temperature transition increases by 9.2^oC in the presence of excess ADP, which is known to bind to the N-terminal domain. The middle transition can be assigned to the Cla and Clb transitions of the C-terminal fragment on the basis of the similarity of ΔH and t"m. In the intact protein Cla and Clb 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.
ISSN: 0022-2836

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