THE THERMAL UNFOLDING OF HEVEIN, A SMALL DISULFIDE-RICH PROTEIN

Differential scanning calorimetry was used to study the thermal unfold ing of hevein, a 43-residue disulfide-rich protein whose three-dimensi onal structure has been determined by X-ray diffraction. In the range pH 2.0-3.7 this process was approximately 75% reversible as judged by repeated scans on the same sample. The ratios of van't Hoff to calorim etric enthalpies were considerably larger than one, suggesting that in termolecular cooperation is involved in the unfolding of this protein. Alternatively, it is possible that the partial irreversibility of thi s process may cause distortions of the endotherm that affect the calcu lation of the van't Hoff enthalpy. Experimental changes in heat capaci ty and enthalpy were compared with those calculated from polar and non polar surface areas buried in the native state. It was found that when the unfolded state is represented as an extended chain without disulf ide cross-links, experimental and calculated parameters agree well. Ho wever, if the unfolded protein is modeled with the presence of disulfi de bridges, the agreement between the two sets of parameters is lost. The entropy change/residue at 112 degrees C is considerably smaller th an the average value for globular proteins, thus suggesting that, as e xpected, disulfide bonds strongly influence the entropy of the unfolde d state of this protein.