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Biochemistry. 1994 Aug 9;33(31):9209-19.

Differential scanning calorimetric study of the thermal unfolding transitions of yeast iso-1 and iso-2 cytochromes c and three composite isozymes.

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  • 1Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.


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.

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