SUBUNIT INTERACTIONS PROVIDE A SIGNIFICANT CONTRIBUTION TO THE STABILITY OF THE DIMERIC 4-ALPHA-HELICAL-BUNDLE PROTEIN ROP

Detailed thermodynamic and spectroscopic studies were carried out on t he ColE1-ROP protein in order to establish a quantitative basis for th e contribution of noncovalent interactions to the stability of four-he lix-bundle proteins. The energetics of both heat- and GdnHCl-induced d enaturation were measured by differential scanning microcalorimetry (D SC) and/or by following the change in circular dichroism in the far-UV range. Sedimentation equilibrium analyses were performed to character ize the state of aggregation of the protein. No intermediate species c ould 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 doubl e-line arrow pointing left and right 2D are DELTAH(D) = 580 +/- 20 kJ. (mol of dimer)-1, DELTAC(p) = 10.3 +/- 1.3 kJ.(mol of dimer)-1.K-1, an d T(m) = 71.0 +/- 0.5-degrees-C. The corresponding Gibbs energy change of unfolding is DELTAG(D)degrees = 71.7 kJ.(mol of dimer)-1 at 25-deg rees-C and pH 6. In the presence of 2.5 M GdnHCl, however, ROP dissoci ates into monomers at elevated temperatures, as the loss of the concen tration dependence of T(m) and the decreased molecular weight demonstr ate. The corresponding transition parameters are DELTAH(D) (2.5 M GdnH Cl) = 130 +/- 10 kJ.(mol of monomer)-1 and T(m) = 51.6 +/- 0.3-degrees -C. Isothermal unfolding studies at 19-degrees-C using GdnHCl as denat urant yielded a Gibbs energy change of unfolding of 22.4 kJ.(mol of mo nomer)-1. This extrapolated value is 38% lower than the corresponding DELTAG(D)degrees 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 int eractions are an important source of stabilization of the native four- helix-bundle structure of ROP.