Abstract
The Escherichia coli mazEF addiction module plays a crucial role in the cell death program that is triggered under various stress conditions. It codes for the toxin MazF and the antitoxin MazE, which interferes with the lethal action of the toxin. To better understand the role of various conformations of MazE in bacterial life, its order-disorder transitions were monitored by differential scanning calorimetry, spectropolarimetry, and fluorimetry. The changes in spectral and thermodynamic properties accompanying MazE dimer denaturation can be described in terms of a compensating reversible process of the partial folding of the unstructured C-terminal half (high mean net charge, low mean hydrophobicity) and monomerization coupled with the partial unfolding of the structured N-terminal half (low mean net charge, high mean hydrophobicity). At pH<or=4.5 and T<50 degrees C, the unstructured polypeptide chains of the MazE dimer fold into (pre)molten globule-like conformations that thermally stabilize the dimeric form of the protein. The simulation based on the thermodynamic and structural information on various addiction modules suggests that both the conformational adaptability of the dimeric antitoxin form (binding to the toxins and DNA) and the reversible transformation to the more flexible monomeric form are essential for the regulation of bacterial cell life and death.
Highlights
The Escherichia coli mazEF addiction module plays a crucial role in the cell death program that is triggered under various stress conditions
We have proved by gel filtration that it exists as a dimer in buffer solutions and that its dimeric state around room temperature remains unchanged under the experimental conditions applied in our studies
Energetics of the MazE Structure—The stability curves (⌬G0 versus temperature) of MazE at pH 7.1 obtained from thermal and urea-induced denaturation are presented in Fig. 6 together with the corresponding enthalpy and entropy contributions
Summary
The expression and purification of MazE have been described previously [46]. MazE solutions for calorimetric and spectroscopic measurements were prepared by diluting the stock solution of MazE in water to the appropriate concentrations in the appropriate buffer solutions. According to the model (Equation 1), one can express an average of a physical property, F (in our case, F is the partial molar enthalpy of the protein, the mean residue ellipticity ([]), or the fluorescence intensity (FL)), in terms of the corresponding contributions FN and FD, which characterize states N and D [47, 59], respectively (Equation 3). Taking into account Equations 1– 4, the observed temperature profiles (melting curves) can be described in terms of the parameters ⌬H0(T1⁄2), ⌬Cp0, and T1⁄2 Their values were obtained from fitting the model function (CD ϭ Equation 3 and DSC ϭ Equation 4) to the experimental temperature profiles using the Levenberg-Marquardt nonlinear 2 regression procedure [63]. ⌬Cp0 accompanying MazE denaturation and dissociation (1/2N2 7 D) was calculated from the corresponding changes in non-polar and polar accessible areas using the expression introduced by Murphy and Freire [65] (Equation 5). The postulates and the details on the numerical procedure are given in the Supplemental Material
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