DIFFERENTIAL SCANNING CALORIMETRIC STUDY OF THE THERMAL UNFOLDING TRANSITIONS OF YEAST ISO-1 AND ISO-2 CYTOCHROMES-C AND 3 COMPOSITE ISOZYMES

The effects of regional sequence differences on the thermodynamic stab ility of a globular protein have been investigated by scanning microca lorimetry. Thermal transitions have been measured for two isozymes of yeast cytochrome c (iso-1-MS and iso-2) and three composite proteins ( Comp1-MS, Comp2-MS, and Comp3-MS) in which amino acid segments are exc hanged between the parental isozymes. There are three main observation s. (1) In the temperature range of the unfolding transitions (40-60 de grees C) the unfolding free energies for the composite proteins are on ly slightly different from those of the the parental isozymes, althoug h in some cases there are large but compensating changes in the transi tional enthalpy and entropy. At lower temperatures (0-30 degrees C), t he Comp1-MS protein which contains only a small amount of iso-2-like s equence is less stable than either of the parental isozymes, despite t he fact that none of the iso-2-specific amino acid side chains impinge s directly on any of the iso-1-specific amino acid side chains. (3) Ch anges in ionization of his 26 appear to be linked to thermal unfolding . Iso-1-MS and Comp1-MS contain a histidine residue at position 26 whi le iso-2 and the other two composites do not. On lowering the pH from pH 6 to 5, both iso-1-MS and Comp1-MS show a decrease in stability (lo wer T-m) within the unfolding transition region (40-60 degrees C), whe reas the stabilities of iso-2, Comp2-MS, and Comp3-MS are essentially unchanged. The thermal unfolding transitions are highly reversible (>9 5%) but mechanistically complex. A moderate dependence of T-m on prote in concentration and the ratio of the van't Hoff enthalpy to the calor imetric enthalpy suggest that thermal unfolding involves the reversibl e association of a significant fraction of the unfolded species, at le ast at elevated protein concentrations.