THERMODYNAMICS OF DENATURATION OF BARSTAR - EVIDENCE FOR COLD DENATURATION AND EVALUATION OF THE INTERACTION WITH GUANIDINE-HYDROCHLORIDE

Isothermal guanidine hydrochloride (GdnHCl)-induced denaturation curve s obtained at 14 different temperatures in the range 275-323 K have be en used in conjunction with thermally-induced denaturation curves obta ined in the presence of 15 different concentrations of GdnHCl to chara cterize the thermodynamics of cold and heat denaturation of barstar. T he Linear free energy model has been used to determine the excess chan ges in free energy, enthalpy, entropy, and heat capacity that occur on denaturation. The stability of barstar in water decreases as the temp erature is decreased from 300 to 273 K. This decrease in stability is not accompanied by a change in structure as monitored by measurement o f the mean residue ellipticities at both 222 and 275 nm. When GdnHCl i s present at concentrations between 1.2 and 2.0 M, the decrease in sta bility with decrease in temperature is however so large that the prote in undergoes cold denaturation. The structural transition accompanying the cold denaturation process has been monitored by measuring the mea n residue ellipticity at 222 nm. The temperature dependence of the cha nge in free energy, obtained in the presence of 10 different concentra tions of GdnHCl in the range 0.2-2.0 M, shows a decrease in stability with a decrease as well as an increase in temperature from 300 K. Valu es of the thermodynamic parameters governing the cold and the heat den aturation of barstar have been obtained with high precision by analysi s of these bell-shaped stability curves. The change in heat capacity a ccompanying the unfolding reaction, Delta C-P, has a value of 1460 +/- 70 cal mol(-1) K-1 in water. The dependencies of the changes in entha lpy, entropy, free energy, and heat capacity on GdnHCl concentration h ave been analyzed on the basis of the linear free energy model. The ch anges in enthalpy (Delta H-i) and entropy (Delta S-i), which occur on preferential binding of GdnHCl to the unfolded state, vis-a-vis the fo lded state, both have a negative value at low temperatures. With an in crease in temperature Delta H-i makes a less favorable contribution, w hile Delta S-i makes a more favorable contribution to the change in fr ee energy (Delta G(i)) due to this interaction, The change in heat cap acity (Delta C-Pi) that occurs on preferential interaction of GdnHCl w ith the unfolded form has a value of only 53 +/- 36 cal mol(-1) K-1 M( -1) The data validate the linear free energy model that is commonly us ed to analyze protein stability.