Detailed thermodynamic and spectroscopic studies were carried out on the ColE1-ROP protein in order to establish a quantitative basis for the contribution of noncovalent interactions to the stability of four-helix-bundle proteins. The energetics of both heat- and GdnHCl-induced denaturation were measured by differential scanning microcalorimetry (DSC) and/or by following the change in circular dichroism in the far-UV range. Sedimentation equilibrium analyses were performed to characterize the state of aggregation of the protein. No intermediate species could be detected during thermal unfolding of the dimer in the absence of GdnHCl. Under these conditions ROP unfolding exhibits a strict two-state behavior. The thermodynamic parameters for the reaction N2<->2D are delta HD = 580 +/- 20 kJ.(mol of dimer)-1, delta Cp = 10.3 +/- 1.3 kJ.(mol of dimer)-1.K-1, and Tm = 71.0 +/- 0.5 degrees C. The corresponding Gibbs energy change of unfolding is delta GD degree = 71.7 kJ.(mol of dimer)-1 at 25 degrees C and pH 6. In the presence of 2.5 M GdnHCl, however, ROP dissociates into monomers at elevated temperatures, as the loss of the concentration dependence of Tm and the decreased molecular weight demonstrate. The corresponding transition parameters are delta HD (2.5 M GdnHCl) = 130 +/- 10 kJ.(mol of monomer)-1 and Tm = 51.6 +/- 0.3 degrees C. Isothermal unfolding studies at 19 degrees C using GdnHCl as denaturant yielded a Gibbs energy change of unfolding of 22.4 kJ.(mol of monomer)-1. This extrapolated value is 38% lower than the corresponding delta GD degree value of 35.85 kJ.(mol of monomer)-1 calculated from thermal unfolding for the monomer in the absence of GdnHCl, where the protein is known to be a dimer. These results suggest that subunit interactions are an important source of stabilization of the native four-helix-bundle structure of ROP. Study holds ProTherm entries: 2887, 11830 Extra Details: additive : EDTA(1 mM), protein stability; four-helix bundle; Rop; Gibbs energy;,thermal unfolding; subunit interactions
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:20 p.m.
|Number of data points||6|
|Proteins||Regulatory protein rop ; Regulatory protein rop|
|Assays/Quantities/Protocols||Experimental Assay: dCp temp:25.0 C ; Experimental Assay: dG ; Experimental Assay: dCp ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHADELYRSCLARFGDDGENL|