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J Mol Biol. 1993 Jul 20;232(2):680-92.

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

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Department of Biology and Biocalorimetry Center, Johns Hopkins University, Baltimore, MD 21218.


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.

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