CONFORMATIONAL STABILITY OF THE ESCHERICHIA-COLI HPR PROTEIN - TEST OF THE LINEAR EXTRAPOLATION METHOD AND A THERMODYNAMIC CHARACTERIZATIONOF COLD DENATURATION

The conformational stability of the histidine-containing phosphocarrie r protein (HPr) from Escherichia coli has been determined using a comb ination of thermal unfolding and urea denaturation experiments. The an alysis of the denaturation data provides a measure of the changes in c onformational free energy, enthalpy, entropy, and heat capacity that a ccompany the equilibrium folding of HPr over a wide range of temperatu re and urea concentrations, In moderate concentrations of urea, HPr un dergoes both high- and low-temperature unfolding, allowing for a relia ble determination of the change in heat capacity for the conformationa l transition, The data are consistent with the linear free energy rela tionship commonly employed to analyze protein denaturation data, even over a relatively large temperature and urea concentration range. Furt hermore, we find that a temperature-independent Delta C-p is adequate to describe HPr stability over the accessible temperature range. Final ly, our data allow us to evaluate the energetics of the urea-protein i nteraction. For HPr, the changes in excess enthalpy and entropy of the denaturant-protein interaction(s) make only minor contributions to th e observed Delta H and Delta S terms, presumably due in some part to t he small size of the HPr protein.