Differential Scanning Calorimetric Study of the Thermal Unfolding Transitions of Yeast Iso-1 and Iso-2 Cytochromes c and Three Composite Isoenzymes

Abstract

The effects of regional sequence differences on the thermodynamic stability of a globular protein have been investigated by scanning microcalorimetry. 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 exchanged between the parental isozymes. There are three main observations. (1) In the temperature range of the unfolding transitions (40-60 degrees C) the unfolding free energies for the composite proteins are only slightly different from those of the parental isozymes, although in some cases there are large but compensating changes in the transitional enthalpy and entropy. At lower temperatures (0-30 degrees C), all the composites are significantly less stable than the two parental proteins. (2) Long-range structural effects are responsible for at least some of the observed differences in stability. For example, in the temperature range of the unfolding transitions (40-60 degrees C), the Comp1-MS protein which contains only a small amount of iso-2-like sequence is less stable than either of the parental isozymes, despite the fact that none of the iso-2-specific amino acid side chains impinges directly on any of the iso-1-specific amino acid side chains. (3) Changes 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 while 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 (lower Tm) within the unfolding transition region (40-60 degrees C), whereas the stabilities of iso-2, Comp2-MS, and Comp3-MS are essentially unchanged. The thermal unfolding transitions are highly reversible (> 95%) but mechanistically complex. A moderate dependence of Tm on protein concentration and the ratio of the van't Hoff enthalpy to the calorimetric enthalpy suggest that thermal unfolding involves the reversible association of a significant fraction of the unfolded species, at least at elevated protein concentrations.

The Role of a Conserved Water Molecule in the Redox-dependent Thermal Stability of Iso-1-cytochrome c

“…Although the unfolding of cytochrome c is generally recognized as a two-state transition, we obtained apparent or van't Hoff enthalpies that are greater than the calorimetric enthalpies for each of the samples in our investigation. This indicates that the unfolding transition of iso-1-cytochrome c may be complicated by reversible intermolecular association of the unfolded state (26). Although the thermal unfolding of cytochrome c deviates from that expected for an ideal two-state process, it is possible to determine the reversible thermodynamic parameters of the protein (21).…”

Nonideality and protein thermal denaturation

“…3 Several lines of evidence are inconsistent with aggrega- tion. First, the calorimetric data are not well fit by a model that permits native-and/or denatured-state dimerization 22 :…”

“…In practice, however, this method is unreliable. [22][23][24][25] Because carboxylate enthalpies of ionization are small, ⌬C p can be determined more precisely by measuring the temperature dependence of ⌬H D at several pH values. Because the calorimetry data were analyzed directly and via van't Hoff analysis, both ⌬H cal and ⌬H vH were used to obtain ⌬C p .…”

“…This discrepancy is observed by others. 22,23,32 At low protein concentrations, ⌬H vH and ⌬H cal are equal at any given pH, suggesting that denaturation is a two-state process. However, at protein concentrations Ͼ130 M, the thermodynamic parameters become concentration dependent ( Figure 5).…”

The role of quaternary interactions on the stability and activity of ascorbate peroxidase

“…The FPLC gel filtration studies demonstrate that at low protein concentrations, the El 12K mutant behaves as a monomer while wild-type APx is primarily a dimer at the lower limit of concentrations, 250 nM, detectable by the FPLC method. The interface mutants are also less stable than wild-type APx, as indi- Ghosaini and coworkers (Ghosaini et al, 1988) (T,,J: This equation can be modified to calculate the differences in AG values of the wild-type protein at a reference temperature, Tret = T,,, vs. the 1 12 mutants at their T,,'s (Tm;,,ut) (Liggins et al, 1994). Substituting Equation 1 into AAC(T,f) = AC,,,(T,f) -AC,,(T,f) yields T,;,,,) -unusual since we might expect the packing of oligomers to be more similar to the packing of the protein interior.…”